Methods for the prevention and the treatment of extracapillary glomerulonephritis

ABSTRACT

The present invention relates to the prevention and the treatment of extracapillary glomerulonephritis such as rapidly progressive glomerulonephritis and collapsing glomerulonephritis.

FIELD OF THE INVENTION

The present invention relates to the prevention and the treatment ofextracapillary glomerulonephritis such as rapidly progressiveglomerulonephritis and collapsing glomerulonephritis.

BACKGROUND OF THE INVENTION

Extracapillary glomerulonephritis is a proliferative glomerulonephritissuch as rapidly progressive glomerulonephritis and collapsingglomerulopathy. The term “extracapillary proliferation” describesproliferation of glomerular epithelial cells, namely podocytes andparietal epithelial cells.

Glomerular injury during crescentic rapidly progressiveglomerulonephritis (RPGN) manifests as a proliferative histologicalpattern with accumulation of inflammatory cells, fibrin andproliferation of intrinsic glomerular cells in Bowman's space(“crescents”) and rapid deterioration of renal function within days ormonths.

Rapidly progressive glomerulonephritis (RPGN) complicating necrotizingcrescentic glomerulonephritis represents the most severe form ofglomerular involvement and can occur in the setting of variousimmunological disorders, including anti-glomerular basement membrane(anti-GBM), ANCA-associated vasculitis, or immune complex diseases likelupus and infectious diseases (1, 2). Strikingly, crescent formationseems to occur downstream to inflammatory injury of the glomerulus in away that is relatively similar whatever the causative immunologicaldisorder. The explanation for this observation might be that thepathogenesis of RPGN is not restricted to the action of inflammatorycells and immune mediators. It has become clear that proliferation ofparietal epithelial cells (3) and podocytes (4) plays a key role increscent formation. Studies in human biopsies (5) and in a mouse model(6) demonstrated that podocytes dysregulated in RPGN, losing theiroriginal cell markers and switching to a proliferative phenotype.Convincing evidence for podocyte involvement in RPGN also came from aseminal study in a murine model where podocyte-specific deletion of theVhl gene resulted in proliferation of podocytes, crescent formation andrapid onset of renal failure (7). The inventors recently demonstratedthat the activation of the epidermal growth factor receptor (EGFR) inpodocytes by de novo expression of the heparin-binding epidermal growthfactor-like growth factor (HB-EGF) also plays a major role in thedevelopment of RPGN (8). Numerous proteins and signaling pathways can beactivated downstream to EGFR activation, including proteins of thesignal transducer and activator of transcription (STAT) family, namelySTAT5 (9) and STAT3 (10). STAT3-SH2 domain can directly bindphosphorylated EGFR on tyrosine 1068 and tyrosine 1086 (11). STAT3 is aknown transducer of signals from growth factors and cytokines and playsimportant roles in development, cell growth, prevention of apoptosis,proliferation and inflammation (12).

Accordingly, there is a need to develop new drugs that will be suitablefor preventing or treating rapidly progressive glomerulonephritis(RPGN). In this way, it has been suggested that characterization of newcompounds for treatment of RPGN may be highly desirable.

Collapsing glomerulopathy (CG) is a different kidney disease and is amorphologic variant of focal segmental glomerulosclerosis (FSGS)characterized by segmental and global collapse of the glomerularcapillaries, marked hypertrophy and hyperplasia of podocytes, and severetubulointerstitial disease (Albaqumi M, Barisoni L. Current views oncollapsing glomerulopathy. J Am Soc Nephrol. 2008 July; 19(7):1276-81.PMID:18287560; Schwimmer J A, Markowitz G S, Valeri A, Appel G B.Collapsing glomerulopathy. Semin Nephrol. 2003 March; 23(2):209-18.PMID:12704581). The pathogenesis of collapsing focal segmentalglomerulosclerosis (FSGS) in patients not infected with HIV is notclear. As with HIV-associated nephropathy, the underlying pathogenicevent appears to be a severe insult to the integrity and biology of theglomerular visceral (podocytes) and parietal epithelial cells. Thisdamage ultimately results in cellular dedifferentiation andproliferation of these glomerular epithelial cells accompanied by aprofound loss of the glomerular filtration barrier function as seen inRPGN. Activation of the STAT family, namely STAT3, has been shown in HIV(human immunodeficiency virus)-associated nephropathy, a common form ofCG (He J C, Husain M, Sunamoto M, D'Agati V D, Klotman M E, Iyengar R,Klotman P E. Nef stimulates proliferation of glomerular podocytesthrough activation of Src-dependent Stat3 and MAPK1,2 pathways. J ClinInvest. 2004 September; 114(5):643-51. PMID:15343382).

Accordingly, the characterization of new compounds for treatment of CGis highly desirable.

MicroARNs (miRNAs) are endogenous small nucleotide single-stranded noncoding RNA that can disrupt protein expression by inducing translationinhibition and mRNA degradation. Recent evidence indicates that miRNAcould have a pivotal role in renal disorders (13). Because STAT3 canactivate the expression of various miRNAs in several proliferativedisorders (14-16) and a study has found a highly conserved STAT3-bindingsite in the promoter region of the miR-17/92 gene in non kidney cell(17), the inventors have studied the expression of miR-92 in STAT3modulation model.

There is no disclosure in the art of the role of miR-92a in rapidlyprogressive glomerulonephritis (RPGN) (or necrotizing crescenticglomerulonephritis) nor in collapsing glomerulopathy (CG), and the useof miR-92a inhibitor compounds in the prevention or treatment of RPGNand CG.

SUMMARY OF THE INVENTION

The present invention relates to miR-92a inhibitor compound for use inthe treatment of extracapillary glomerulonephritis in a subject in needthereof.

Particularly, the present invention relates to miR-92a inhibitorcompound for use in the treatment of rapidly progressiveglomerulonephritis (RPGN) and collapsing glomerulopathy in a subject inneed thereof.

The present invention also relates to a method of identifying a subjecthaving or at risk of having or developing extracapillaryglomerulonephritis, comprising a step of measuring in a sample obtainedfrom said subject the expression level of miR-92a.

Particularly, the present invention relates to a method of identifying asubject having or at risk of having or developing rapidly progressiveglomerulonephritis (RPGN) and collapsing glomerulopathy (CG), comprisinga step of measuring in a sample obtained from said subject theexpression level of miR-92a.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the inventor investigated pathologicaldedifferentiation of glomerular cells and microRNAs deregulation inrapidly progressive glomerulonephritis (RPGN), an extracapillaryglomerulonephritis.

The inventors found that microRNA-92a (miR-92a) expression in diseasedglomeruli is upregulated in experimental RPGN. The inventorsdemonstrated that EGFR, Interleukine-6 (IL-6) and STAT3 cascadecontrolled de novo expression of miR-92a in primary podocytes.Furthermore, upregulation of miR-92a glomerular expression during RPGNwas found to be abrogated in vivo by EGFR kinase inhibition or podocytespecific deletion of Stat3 in mice.

The inventors also demonstrated that in vivo silencing of miR-92a usingantagomiR (anti-miR-92a) strategy de-repressed CDK-inhibitor p57expression in podocytes and prevented podocyte proliferation, glomerulardemolition and renal failure. The inventors also observed thatcritically ill patients with RPGN had increased phospho-STAT3 andmiRNA-92a glomerular expression compared to normal kidneys. and inkidney biopsies from patients diagnosed with or necrotizing crescenticglomerulonephritis of various cause, including systemic lupuserthematosus (SLE), anti-neutrophil cytoplasmic autoantibody(ANCA)-associated vasculitides and Goodpasture syndrome. miR-92aexpression detected by in situ hybridization and RT-PCR in diseasedglomeruli is a specific feature of necrotizing crescenticglomerulonephritis and was not found in normal kidneys and in kidneybiopsies from individuals diagnosed other glomerular proteinuricdiseases with no extracapillary cell proliferation such as minimalchange disease, membranous nephropathy and diabetic nephropathy. It isanticipated that miR-92a is involved in any other kind of glomerulardisease with extracapillary cell proliferation, including collapsingglomerulopathy.

The present invention demonstrates the implication of miR-92a inextracapillary glomerulonephritis and RPGN development with asignificant pathogenic, diagnostic, and/or therapeutic implications.

Therapeutic Methods and Uses

Accordingly, the present invention relates to a miR-92a inhibitorcompound for use in the prevention and treatment of extracapillaryglomerulonaphritis in a subject in need thereof.

In a particular embodiment, the present invention relates to a miR-92ainhibitor compound for use in the prevention and treatment of rapidlyprogressive glomerulonephritis (RPGN) in a subject in need thereof.

In a particular embodiment, the present invention relates to a miR-92ainhibitor compound for use in the prevention and treatment of collapsingglomerulopathy (CG) in a subject in need thereof.

As used herein, the term “miR-92a” has its general meaning in the artand refers to the miR-92a sequence available from the data basehttp://microrna.sanger.ac.uk/sequences/under the miRBase Accessionnumbers MI0000093 (hsa-mir-92a-1, SEQ ID NO: 1), MIMAT0004507(hsa-miR-92a-1-5p, SEQ ID NO: 2), MIMAT0000092 (hsa-miR-92a-3p, SEQ IDNO: 3), MI0000094 (hsa-mir-92a-2, SEQ ID NO: 4), MIMAT0004508(hsa-miR-92a-2-5p, SEQ ID NO: 5), MIMAT0000092 (hsa-miR-92a-3p, SEQ IDNO: 6).

As used herein, the term “subject” denotes a mammal. In a preferredembodiment of the invention, a subject according to the invention refersto any subject (preferably human) afflicted with or susceptible to beafflicted with extracapillary glomerulonephritis, particularly, rapidlyprogressive glomerulonephritis (RPGN) or collapsing glomerulopathy (CG).

As used herein, the term “extracapillary glomerulonephritis” is used todesignate the cellular proliferation that occupies the Bowman's space.The term “extracapillary” indicates that cell proliferation occursoutside of the capillary tuft. Extracapillary proliferation involvesglomerular epithelial cells, namely podocytes and parietal epithelialcells.

As used herein, the term “rapidly progressive glomerulonephritis” or“RPGN” has its general meaning in the art and refers to crescenticrapidly progressive glomerulonephritis, the glomerular injury thatmanifests as a proliferative histological pattern with accumulation ofinflammatory cells and proliferation of intrinsic glomerular cells inBowman's space (“crescents”) and rapid deterioration of renal function.The term “Rapidly Progressive Glomerulonephritis” relates to crescenticglomerulonephritis or necrotizing crescentic glomerulonephritis orextracapillary glomerulonephritis (Jenette J C and Thomas D B.Crescentic glomerulonephritis. Nephrol Dial Transplant. 2001; 16 Suppl6:80-2; Moeller M J, Soofi A, Hartmann I, et al. Podocytes populatecellular crescents in a murine model of inflammatory glomerulonephritis.J Am Soc Nephrol 2004; 15:61-67; Tarzi R M, Cook H T, Pusey C D.Crescentic glomerulonephritis: new aspects of pathogenesis. SeminNephrol. 2011 July; 31(4):361-8; King S K, Jeansson M, Quaggin S E etal. New insights into the pathogenesis of cellular crescents. CurrentOpinion in Nephrology and Hypertension 2011, 20:258-262; Robert M.Kliegman, MD, Bonita M.D. Stanton, MD, Joseph St. Geme, Nina Schor andRichard E. Behrman, MD. Chapter 510—Rapidly Progressive (Crescentic)Glomerulonephritis. Nelson Textbook of Pediatrics, 19th Edition—SaundersTitle, ISBN: 978-1-4377-0755-7).

RPGN can be primary or secondary. Secondary forms occur in any form ofsevere glomerulonephritis including membranoproliferative GN, IgAnephropathy, post infectious GN, anti-neutrophil cytoplasmicautoantibody (ANCA)-associated vasculitides, and systemic lupuserythematous (SLE). RPGN can be of various etiologies including stageIII and IV lupus nephritis, microscopic polyangiitis (MPA) andgranulomatosis with polyangiitis (GPA).

As used herein, the term “collapsing glomerulopathy” or “CG” has itsgeneral meaning in the art anf refers to a distinct entity, so calledcollapsing focal segmental glomerulosclerosis (FSGS), also involvesextracapillary proliferation and marked dysregulation of the quiescentpodocyte phenotype (Bariety J, Nochy D, Mandet C, Jacquot C, Glotz D,Meyrier A: Podocytes undergo phenotypic changes and expressmacrophagic-associated markers in idiopathic collapsing glomerulopathy.Kidney Int 53: 918-925, 1998. Srivastava T, Garola R E, Singh H K:Cell-cycle regulatory proteins in the podocyte in collapsingglomerulopathy in children. Kidney Int 70: 529-535, 2006). Diseasedpodocytes exhibit a loss and gain of markers of differentiation andproliferation, respectively. Recent studies indicate that parietalepithelial cells also may be recruited into the viscerally locatedproliferative lesion (Dijkman H B, Weening J J, Smeets B, Verrijp K C,van Kuppevelt T H, Assmann K K, Steenbergen E J, Wetzels J:Proliferating cells in HIV and pamidronate-associated collapsing focalsegmental glomerulosclerosis are parietal epithelial cells. Kidney Int70: 338-344, 2006).

Collapsing glomerulopathy (CG) can be primary or secondary (Albaqumi M,Barisoni L. Current views on collapsing glomerulopathy. J Am SocNephrol. 2008 July; 19(7):1276-81. PMID:18287560). Secondary forms canbe associated to HIV infection or other viral infections (such ashepatitis C virus infection, and parvovirus), drug addiction,pamidronate, systemic lupus erythematosus-like disorder and multiplemyeloma or can be favored by genetic background.

As used herein, the term “miR-92a inhibitor compound” refers to anycompound able to prevent the action of miR-92a. The miR-92a inhibitorcompound of the present invention is a compound that inhibits or reducesthe activity of miR-92a. However, decreasing and/or reducing theactivity of miR-92a can also be obtained by inhibiting miR-92aexpression. The term “inhibiting miR-92a expression” means that theproduction of miR-92a in the podocytes after treatment is less than theamount produced prior to treatment. One skilled in the art can readilydetermine whether miR-92a expression has been inhibited in a kidney orpodocytes, using for example the techniques for determining miRNAtranscript level.

In a particular embodiment, miR-92a inhibitor compound of the inventionis a compound such as nucleic acid that hybridizes with miR-92a orhaving sequence complementarity to that of miR-92a. In a particularembodiment, miR-92a inhibitor compound of the invention is a compoundsuch as nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% sequence complementarity to that of miR-92a.

Suitable miR-92a inhibitor compounds include double-stranded RNA (suchas short- or small-interfering RNA or “siRNA”), antagomirs, antisensenucleic acids, and enzymatic RNA molecules such as ribozymes. Each ofthese compounds can be targeted to a given miRNA and destroy or inducethe destruction of the target miRNA. For example, expression of a givenmiRNA can be inhibited by inducing RNA interference of the miRNA with anisolated double-stranded RNA (“dsRNA”) molecule which has at least 90%,for example 95%, 96%, 97%, 98%, 99% or 100%, sequence homology with atleast a portion of the miRNA. In a preferred embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA”.

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence whichis substantially identical to a nucleic acid sequence contained withinthe target miRNA.

As used herein, a nucleic acid sequence in a siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area. The siRNA canalso be altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, or modifications that make the siRNA resistantto nuclease digestion, or the substitution of one or more nucleotides inthe siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3 overhang. As usedherein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in oneembodiment, the siRNA comprises at least one 3′ overhang of 1 to about 6nucleotides (which includes ribonucleotides or deoxyribonucleotides) inlength, preferably from 1 to about 5 nucleotides in length, morepreferably from 1 to about 4 nucleotides in length, and particularlypreferably from about 2 to about 4 nucleotides in length. In a preferredembodiment, the 3′ overhang is present on both strands of the siRNA, andis 2 nucleotides in length. For example, each strand of the siRNA cancomprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid(“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as describedabove. Exemplary methods for producing and testing dsRNA or siRNAmolecules are described in U.S. published patent application2002/0173478 to Gewirtz and in U.S. published patent application2004/0018176 to Reich et al., the entire disclosures of which are hereinincorporated by reference.

Expression of a given miRNA can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA orRNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequencecomplementary to a contiguous nucleic acid sequence in a miRNA.Preferably, the antisense nucleic acid comprises a nucleic acid sequencethat is 50-100% complementary, more preferably 75-100% complementary,and most preferably 95-100% complementary to a contiguous nucleic acidsequence in an miRNA. Nucleic acid sequences for the miRNAs are providedin Table A. Without wishing to be bound by any theory, it is believedthat the antisense nucleic acids activate RNase H or some other cellularnuclease that digests the miRNA/antisense nucleic acid duplex.

In a preferred embodiment the inhibitor is an antagomir and/or anantisense oligonucleotide.

The term “antagomir” or “antagomiR-92a” as used herein refers to achemically engineered small RNA that is used to silence miR-92a. Theantagomir is complementary to the specific miRNA target with eithermis-pairing or some sort of base modification. Antagomirs may alsoinclude some sort of modification to make them more resistant todegradation. In a preferred embodiment the antagomir is a chemicallyengineered cholesterol-conjugated single-stranded RNA analogue.

Inhibition of miR-92a can also be achieved with antisense 2′-O-methyl(2′-O-Me) oligoribonucleotides, 2′-O-methoxyethyl (2′-O-MOE),phosphorothioates, locked nucleic acid (LNA), morpholino oligomers or byuse of lentivirally or adenovirally expressed antagomirs (Stenvang andKauppinen (2008), Expert Opin. Biol. Ther. 8(1):59-81). Furthermore, MOE(2′-O-methoxyethyl phosphorothioate) or LNA (locked nucleic acid (LNA)phosphorothioate chemistry)-modification of single-stranded RNAanalogous can be used to inhibit miRNA activity.

Antisense nucleic acids can also contain modifications of the nucleicacid backbone or of the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators such as acridine orthe inclusion of one or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed below. Exemplary methods for producing and testing are withinthe skill in the art; see, e.g., Stein and Cheng (1993), Science261:1004 and U.S. Pat. No. 5,849,902 to Woolf et al., the entiredisclosures of which are herein incorporated by reference.

Expression of a given miRNA can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miRNA, andwhich is able to specifically cleave the miRNA. Preferably, theenzymatic nucleic acid substrate binding region is 50-100%complementary, more preferably 75-100% complementary, and mostpreferably 95-100% complementary to a contiguous nucleic acid sequencein a miRNA. The enzymatic nucleic acids can also comprise modificationsat the base, sugar, and/or phosphate groups. An exemplary enzymaticnucleic acid for use in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed below. Exemplary methods for producing and testing dsRNA orsiRNA molecules are described in Werner and Uhlenbeck (1995), Nucl.Acids Res. 23:2092-96; Hammann et al. (1999), Antisense and Nucleic AcidDrug Dev. 9:25-31; and U.S. Pat. No. 4,987,071 to Cech et al, the entiredisclosures of which are herein incorporated by reference.

The miR-92a inhibitor compound of the invention can be obtained using anumber of standard techniques. For example the miR-92a inhibitorcompound of the invention can be chemically synthesized or recombinantlyproduced using methods known in the art. Typically, miR-92a inhibitorcompound of the invention are chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. Commercial suppliers of synthetic RNA molecules orsynthesis reagents include, e.g., Proligo (Hamburg, Germany), DharmaconResearch (Lafayette, Colo., USA), Pierce Chemical (part of PerbioScience, Rockford, Ill., USA), Glen Research (Sterling, Va., USA),ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

In some embodiments, of the invention, a synthetic miR-92a inhibitorcompound of the invention contains one or more design elements. Thesedesign elements include, but are not limited to: (i) a replacement groupfor the phosphate or hydroxyl of the nucleotide at the 5′ terminus ofthe complementary region; (ii) one or more sugar modifications. Incertain embodiments, a synthetic miR-92a inhibitor compound of theinvention has a nucleotide at its 5′ end of the complementary region inwhich the phosphate and/or hydroxyl group has been replaced with anotherchemical group (referred to as the “replacement design”). In some cases,the phosphate group is replaced, while in others, the hydroxyl group hasbeen replaced. In particular embodiments, the replacement group isbiotin, an amine group, a lower alkylamine group, an acetyl group,2′O-Me (2′oxygen-methyl), DMTO (4,4′-dimethoxytrityl with oxygen),fluorescein, a thiol, or acridine, though other replacement groups arewell known to those of skill in the art and can be used as well. Inparticular embodiments, the sugar modification is a 2′O-Me modification.In further embodiments, there is one or more sugar modifications in thefirst or last 2 to 4 residues of the complementary region or the firstor last 4 to 6 residues of the complementary region.

In a particular embodiment, the miR-92a inhibitor compound of theinvention is resistant to degradation by nucleases. One skilled in theart can readily synthesize nucleic acids which are nuclease resistant,for example by incorporating one or more ribonucleotides that aremodified at the 2′-position into the miRNAs. Suitable 2′-modifiedribonucleotides include those modified at the 2′-position with fluoro,amino, alkyl, alkoxy, and O-allyl.

The present invention also relates to a vector comprising a miR-92ainhibitor compound according to the invention for use in the preventionand treatment of extracapillary glomerulonephritis, rapidly progressiveglomerulonephritis (RPGN) and collapsing glomerulopathy (CG).

Alternatively, the miR-92a inhibitor compound of the invention can beexpressed from recombinant circular or linear DNA plasmids using anysuitable promoter. Suitable promoters for expressing RNA from a plasmidinclude, e.g., the U6 or HI RNA pol III promoter sequences, or thecytomegalovirus promoters. Selection of other suitable promoters iswithin the skill in the art. The recombinant plasmids of the inventioncan also comprise inducible or regulatable promoters for expression ofthe miR-92a inhibitor compound of the invention in podocytes.

The miR-92a inhibitor compound of the invention that is expressed fromrecombinant plasmids can be isolated from cultured cell expressionsystems by standard techniques. The miR-92a inhibitor compound of theinvention which is expressed from recombinant plasmids can also bedelivered to, and expressed directly in, the podocytes. The use ofrecombinant plasmids to deliver the miR-92a inhibitor compound of theinvention to podocytes is discussed in more detail below.

The miR-92a inhibitor compound of the invention can be expressed from aseparate recombinant plasmid, or can be expressed from a uniquerecombinant plasmid. Preferably, the miR-92a inhibitor compound of theinvention is expressed as the nucleic acid precursor molecules from asingle plasmid, and the precursor molecules are processed into thefunctional miR-92a inhibitor compound by a suitable processing system,including processing systems extant within podocytes. Other suitableprocessing systems include, e.g., the in vitro Drosophila cell lysatesystem as described in U.S. published application 2002/0086356 to Tuschlet al. and the E. coli RNAse III system described in U.S. publishedpatent application 2004/0014113 to Yang et al., the entire disclosuresof which are herein incorporated by reference.

Selection of plasmids suitable for expressing the miR-92a inhibitorcompound of the invention, methods for inserting nucleic acid sequencesinto the plasmid to express the gene products, and methods of deliveringthe recombinant plasmid to the cells of interest are within the skill inthe art. See, for example, Zeng et al. (2002), Molecular Cell9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp etal. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat.Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958;Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002),Nat. Biotechnol. 20:505-508, the entire disclosures of which are hereinincorporated by reference.

In one embodiment, a plasmid expressing the miR-92a inhibitor compoundof the invention comprises a sequence encoding a miR-92a inhibitorcompound precursor under the control of the CMV intermediate earlypromoter. As used herein, “under the control” of a promoter means thatthe nucleic acid sequences are located 3′ of the promoter, so that thepromoter can initiate transcription of the miR-92a inhibitor compoundcoding sequences.

The miR-92a inhibitor compound of the invention can also be expressedfrom recombinant viral vectors. It is contemplated that the miR-92ainhibitor compound of the invention can be expressed from separaterecombinant viral vectors, or from a unique viral vector. The miR-92ainhibitor compound expressed from the recombinant viral vectors caneither be isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in podocytes. The use ofrecombinant viral vectors to deliver the miR-92a inhibitor compound topodocytes is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR-92a inhibitor compound of the invention and anysuitable promoter for expressing the miR-92a inhibitor compoundsequences. Suitable promoters include, for example, the U6 or HI RNA polIII promoter sequences, or the cytomegalovirus promoters. Selection ofother suitable promoters is within the skill in the art. The recombinantviral vectors of the invention can also comprise inducible orregulatable promoters for expression of the miR-92a inhibitor compoundin podocytes.

Any viral vector capable of accepting the coding sequences for themiR-92a inhibitor compound of the invention can be used; for example,vectors derived from adenovirus (AV); adenoassociated virus (AAV);retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemiavirus); herpes virus, and the like. The tropism of the viral vectors canbe modified by pseudotyping the vectors with envelope proteins or othersurface antigens from other viruses, or by substituting different viralcapsid proteins, as appropriate. For example, lentiviral vectors of theinvention can be pseudotyped with surface proteins from vesicularstomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectorsof the invention can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes. For example,an AAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ. E. et al. (2002), J Virol 76:791801, the entire disclosure of whichis herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingsaid miR-92a inhibitor compound of the invention into the vector,methods of delivering the viral vector to the cells of interest, andrecovery of the expressed miR-92a inhibitor compound products are withinthe skill in the art. See, for example, Dornburg (1995), Gene Therap.2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therap. 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. A suitable AVvector for expressing the miR-92a inhibitor compound of the invention, amethod for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia et al.(2002), Nat. Biotech. 20:1006-1010, the entire disclosure of which isherein incorporated by reference. Suitable AAV vectors for expressingthe miR-92a inhibitor compound of the invention, methods forconstructing the recombinant AAV vector, and methods for delivering thevectors into target cells are described in Samulski et al. (1987), J.Virol. 61:3096-3101; Fisher et al. (1996), J. Virol., 70:520-532;Samulski et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479;U.S. Pat. No. 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.Preferably, the miR-92a inhibitor compound of the invention is expressedfrom a single recombinant AAV vector comprising the CMV intermediateearly promoter.

In one embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR-92a inhibitor compoundprecursor in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter. As used herein, “in operableconnection with a polyT termination sequence” means that the nucleicacid sequences encoding the sense or antisense strands are immediatelyadjacent to the polyT termination signal in the 5′ direction. Duringtranscription of the miR-92a inhibitor compound sequences from thevector, the polyT termination signals act to terminate transcription.

The miR-92a inhibitor compound can be administered to a subject by anymeans suitable for delivering these compounds to kidney or podocytes ofthe subject. For example, the miR-92a inhibitor compound can beadministered by methods suitable to transfect cells of the subject withthese compounds, or with nucleic acids comprising sequences encodingthese compounds. Preferably, the cells are transfected with a plasmid orviral vector comprising sequences encoding at least one miR-92ainhibitor compound.

The miR-92a inhibitor compound can be administered to a subject by anysuitable enteral or parenteral administration route. Suitable enteraladministration routes for the present methods include, e.g., oral,rectal, or intranasal delivery. Suitable parenteral administrationroutes include, e.g., intravascular administration (e.g., intravenousbolus injection, intravenous infusion, intra-arterial bolus injection,intra-arterial infusion and catheter instillation into the vasculature);peri- and intra-tissue injection (e.g., intra-retinal injection, orsubretinal injection); subcutaneous injection or deposition, includingsubcutaneous infusion (such as by osmotic pumps); direct application tothe tissue of interest, for example by a catheter or other placementdevice (e.g., a retinal pellet or a suppository or an implant comprisinga porous, non-porous, or gelatinous material); and inhalation. Preferredadministration routes are injection, infusion and direct injection intothe kidney tissue.

In the present methods, a miR-92a inhibitor compound can be administeredto the subject either as naked RNA, in combination with a deliveryreagent, or as a nucleic acid (e.g., a recombinant plasmid or viralvector) comprising sequences that express the miR-92a inhibitorcompound. Suitable delivery reagents include, e.g, the Minis Transit TKOlipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations(e.g., polylysine), and liposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR-92a inhibitor compounds, and techniques for delivering suchplasmids and vectors to podocytes, are discussed above.

In a preferred embodiment, liposomes are used to deliver a miR-92ainhibitor compound (or nucleic acids comprising sequences encoding them)to a subject. Liposomes can also increase the blood half-life of thegene products or nucleic acids. Liposomes suitable for use in theinvention can be formed from standard vesicle-forming lipids, whichgenerally include neutral or negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of factors such as the desired liposome size andhalf-life of the liposomes in the blood stream.

A variety of methods are known for preparing liposomes, for example, asdescribed in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; andU.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, theentire disclosures of which are herein incorporated by reference. Theliposomes for use in the present methods can comprise a ligand moleculethat targets the liposome to podocytes. Ligands which bind to receptorsprevalent in podocytes, such as monoclonal antibodies that bind topodocytes antigens, are preferred. The liposomes for use in the presentmethods can also be modified so as to avoid clearance by the mononuclearmacrophage system (“MMS”) and reticuloendothelial system (“RES”). Suchmodified liposomes have opsonization-inhibition moieties on the surfaceor incorporated into the liposome structure. In a particularly preferredembodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference. Opsonization inhibiting moieties suitable for modifyingliposomes are preferably water-soluble polymers with a number-averagemolecular weight from about 500 to about 40,000 daltons, and morepreferably from about 2,000 to about 20,000 daltons. Such polymersinclude polyethylene glycol (PEG) or polypropylene glycol (PPG)derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone;linear, branched, or dendrimeric polyamidoamines; polyacrylic acids;polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylicor amino groups are chemically linked, as well as gangliosides, such asganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, orderivatives thereof, are also suitable. In addition, the opsonizationinhibiting polymer can be a block copolymer of PEG and either apolyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, orpolynucleotide. The opsonization inhibiting polymers can also be naturalpolysaccharides containing amino acids or carboxylic acids, e.g.,galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid,pectic acid, neuraminic acid, alginic acid, carrageenan; animatedpolysaccharides or oligosaccharides (linear or branched); orcarboxylated polysaccharides or oligosaccharides, e.g., reacted withderivatives of carbonic acids with resultant linking of carboxylicgroups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes”.

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive animation usingNa(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects will efficiently accumulate these liposomes; see Gabizon, et al.(1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, thereduced uptake by the RES lowers the toxicity of stealth liposomes bypreventing significant accumulation of the liposomes in the liver andspleen. Thus, liposomes that are modified with opsonization-inhibitionmoieties are particularly suited to deliver the miR-92a inhibitorcompounds (or nucleic acids comprising sequences encoding them) topodocytes.

One skilled in the art can readily determine a therapeutically effectiveamount of said compound to be administered to a given subject, by takinginto account factors such as the size and weight of the subject; theextent of disease penetration; the age, health and sex of the subject;the route of administration; and whether the administration is regionalor systemic. An effective amount of said compound can be based on theapproximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of thecompound is administered to a subject can range from about 5-10000micrograms/kg of body weight, and is preferably between about 5-3000micrograms/kg of body weight, and is preferably between about 700-1000micrograms/kg of body weight, and is more preferably greater than about1000 micrograms/kg of body weight. One skilled in the art can alsoreadily determine an appropriate dosage regimen for the administrationof the compound to a given subject. For example, the compound can beadministered to the subject once (e.g., as a single injection ordeposition).

In another embodiment, the present invention relates to a method ofpreventing or treating extracapillary glomerulonephritis in a subject inneed thereof, comprising the step of administering to said subject amiR-92a inhibitor compound.

Pharmaceutical Compositions

The miR-92a inhibitor compound of the invention may be used or preparedin a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a miR-92a inhibitor compound and a pharmaceutical acceptablecarrier for use in the prevention and treatment of extracapillaryglomerulonephritis in a subject in need thereof.

In a particular embodiment, the invention relates to a pharmaceuticalcomposition comprising a miR-92a inhibitor compound and a pharmaceuticalacceptable carrier for use in the prevention and treatment of rapidlyprogressive glomerulonephritis (RPGN) and collapsing glomerulopathy (CG)in a subject in need thereof.

The miR-92a inhibitor compounds of the invention are preferablyformulated as pharmaceutical compositions, prior to administering to apatient, according to techniques known in the art. Pharmaceuticalcompositions of the present invention are characterized as being atleast sterile and pyrogen-free. As used herein, “pharmaceuticalformulations” include formulations for human and veterinary use. Methodsfor preparing pharmaceutical compositions of the invention are withinthe skill in the art, for example as described in Remington'sPharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa.(1985), the entire disclosure of which is herein incorporated byreference.

The present pharmaceutical formulations comprise miR-92a inhibitorcompound (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a pharmaceutically-acceptable carrier. Thepharmaceutical formulations of the invention can also comprise miR-92ainhibitor compound which are encapsulated by liposomes and apharmaceutically-acceptable carrier. Preferredpharmaceutically-acceptable carriers are water, buffered water, normalsaline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the miR-92a inhibitor compound. A pharmaceuticalcomposition for aerosol (inhalational) administration can comprise0.01-20% by weight, preferably 1%-10% by weight, of the miR-92ainhibitor compound encapsulated in a liposome as described above, and apropellant. A carrier can also be included as desired; e.g., lecithinfor intranasal delivery.

Pharmaceutical compositions of the invention may include any furtheragent which is used in the prevention or treatment of extracapillaryglomerulonephritis, rapidly progressive glomerulonephritis (RPGN) orcollapsing glomerulopathy (CG).

For example, the anti-RPGN therapy may include cyclophosphamide,plasmapheresis, anti-CD20 antibody, mycophenolate mofetil andcorticosteroids such as methylprednisolone or prednisone.

For example, the anti-CG therapy may include steroids or cyclosporine,angiotensin converting enzyme inhibitors and/or angiotensin II receptorblockers, anti-HIV therapy, lipid lowering agents and mycophenolatemofetil.

In one embodiment, said additional active agents may be contained in thesame composition or administrated separately.

In another embodiment, the pharmaceutical composition of the inventionrelates to combined preparation for simultaneous, separate or sequentialuse in the prevention and treatment of rapidly progressiveglomerulonephritis (RPGN).

The invention also provides kits comprising the miR-92a inhibitorcompound of the invention. Kits containing the miR-92a inhibitorcompound of the invention find use in therapeutic methods.

Diagnostics Methods

A further aspect of the invention relates to a method of identifying asubject having or at risk of having or developing extracapillaryglomerulonephritis, comprising a step of measuring in a sample obtainedfrom said subject the expression level of miR-92a.

In a particular embodiment, the present invention relates to a method ofidentifying a subject having or at risk of having or developing rapidlyprogressive glomerulonephritis (RPGN), comprising a step of measuring ina sample obtained from said subject the expression level of miR-92a.

In a particular embodiment, the present invention relates to a method ofidentifying a subject having or at risk of having or developingcollapsing glomerulopathy (CG), comprising a step of measuring in asample obtained from said subject the expression level of miR-92a.

The method of the invention may further comprise a step consisting ofcomparing the expression level of miR-92a in the sample with a control,wherein detecting differential in the expression level of the miR-92abetween the sample and the control is indicative of subject having or atrisk of having or developing an extracapillary glomerulonephritis, arapidly progressive glomerulonephritis (RPGN) or a collapsingglomerulopathy (CG).

In one embodiment, the control may consist in sample associated with ahealthy subject not afflicted with extracapillary glomerulonephritis,not afflicted with rapidly progressive glomerulonephritis (RPGN) and notafflicted with collapsing glomerulopathy (CG) as a negative control.Accordingly, a higher expression level of miR-92a in the sample than thecontrol is indicative of a subject having or at risk of having ordeveloping an extracapillary glomerulonephritis, a rapidly progressiveglomerulonephritis (RPGN) or a collapsing glomerulopathy (CG), and alower or equal expression level of miR-92a in the sample than thecontrol is indicative of a subject not having or not at risk of havingor developing an extracapillary glomerulonephritis, a rapidlyprogressive glomerulonephritis (RPGN) or a collapsing glomerulopathy(CG).

In another embodiment, the control may consist in sample associated witha subject afflicted with extracapillary glomerulonephritis, rapidlyprogressive glomerulonephritis (RPGN) or collapsing glomerulopathy (CG)as a positive control. Accordingly, a higher or equal expression levelof miR-92a in the sample than the control is indicative of a subjecthaving or at risk of having or developing an extracapillaryglomerulonephritis, a rapidly progressive glomerulonephritis (RPGN) or acollapsing glomerulopathy (CG), and a lower expression level of miR-92ain the sample than the control is indicative of a subject not having ornot at risk of having or developing an extracapillaryglomerulonephritis, a rapidly progressive glomerulonephritis (RPGN) or acollapsing glomerulopathy (CG).

According to the invention, measuring the expression level of themiR-92a of the invention in the sample obtained from the subject can beperformed by a variety of techniques.

For example the nucleic acid contained in the samples (e.g., cell ortissue prepared from the subject) is first extracted according tostandard methods, for example using lytic enzymes or chemical solutionsor extracted by nucleic-acid-binding resins following the manufacturer'sinstructions. Conventional methods and reagents for isolating RNA from asample comprise High Pure miRNA Isolation Kit (Roche), Trizol(Invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction,PureLink™ miRNA isolation kit (Invitrogen), PureLink Micro-to-Midi TotalRNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit(Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroformextraction, TCA/acetone precipitation, ethanol precipitation, Columnpurification, Silica gel membrane purification, PureYield™ RNA Midiprep(Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System(Promega), SV Total RNA Isolation (Promega), geneMAG-RNA/DNA kit(Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNA™Kit (Ambion), Poly(A)Purist™ Kit (Ambion) and any other methods,commercially available or not, known to the skilled person.

The expression level of miR-92a in the sample may be determined by anysuitable method. Any reliable method for measuring the level or amountof miRNA in a sample may be used. Generally, miR-92a can be detected andquantified from a sample (including fractions thereof), such as samplesof isolated RNA by various methods known for mRNA, including, forexample, amplification-based methods (e.g., Polymerase Chain Reaction(PCR), Real-Time Polymerase Chain Reaction (RT-PCR), QuantitativePolymerase Chain Reaction (qPCR), rolling circle amplification, etc.),hybridization-based methods (e.g., hybridization arrays (e.g.,microarrays), NanoString analysis, Northern Blot analysis, branched DNA(bDNA) signal amplification, in situ hybridization, etc.), andsequencing-based methods (e.g., next-generation sequencing methods, forexample, using the Illumina or IonTorrent platforms). Other exemplarytechniques include ribonuclease protection assay (RPA) and massspectroscopy.

In some embodiments, RNA is converted to DNA (cDNA) prior to analysis.cDNA can be generated by reverse transcription of isolated miRNA usingconventional techniques. miRNA reverse transcription kits are known andcommercially available. Examples of suitable kits include, but are notlimited to the mirVana TaqMan® miRNA transcription kit (Ambion, Austin,Tex.), and the TaqMan® miRNA transcription kit (Applied Biosystems,Foster City, Calif.). Universal primers, or specific primers, includingmiRNA-specific stem-loop primers, are known and commercially available,for example, from Applied Biosystems. In some embodiments, miRNA isamplified prior to measurement. In some embodiments, the expressionlevel of miRNA is measured during the amplification process. In someembodiments, the expression level of miRNA is not amplified prior tomeasurement. Some exemplary methods suitable for determining theexpression level of miRNA in a sample are described in greaterhereinafter. These methods are provided by way of illustration only, andit will be apparent to a skilled person that other suitable methods maylikewise be used.

Many amplification-based methods exist for detecting the expressionlevel of miRNA nucleic acid sequences, including, but not limited to,PCR, RT-PCR, qPCR, and rolling circle amplification. Otheramplification-based techniques include, for example, ligase chainreaction (LCR), multiplex ligatable probe amplification, in vitrotranscription (IVT), strand displacement amplification (SDA),transcription-mediated amplification (TMA), nucleic acid sequence basedamplification (NASBA), RNA (Eberwine) amplification, and other methodsthat are known to persons skilled in the art. A typical PCR reactionincludes multiple steps, or cycles, that selectively amplify targetnucleic acid species: a denaturing step, in which a target nucleic acidis denatured; an annealing step, in which a set of PCR primers (i.e.,forward and reverse primers) anneal to complementary DNA strands, and anelongation step, in which a thermostable DNA polymerase elongates theprimers. By repeating these steps multiple times, a DNA fragment isamplified to produce an amplicon, corresponding to the target sequence.Typical PCR reactions include 20 or more cycles of denaturation,annealing, and elongation. In many cases, the annealing and elongationsteps can be performed concurrently, in which case the cycle containsonly two steps. A reverse transcription reaction (which produces a cDNAsequence having complementarity to a miRNA) may be performed prior toPCR amplification. Reverse transcription reactions include the use of,e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.Kits for quantitative real time PCR of miRNA are known, and arecommercially available. Examples of suitable kits include, but are notlimited to, the TaqMan® miRNA Assay (Applied Biosystems) and themirVana™ qRT-PCR miRNA detection kit (Ambion). The miRNA can be ligatedto a single stranded oligonucleotide containing universal primersequences, a polyadenylated sequence, or adaptor sequence prior toreverse transcriptase and amplified using a primer complementary to theuniversal primer sequence, poly(T) primer, or primer comprising asequence that is complementary to the adaptor sequence. In someembodiments, custom qRT-PCR assays can be developed for determination ofmiRNA levels. Custom qRT-PCR assays to measure miRNAs in a sample can bedeveloped using, for example, methods that involve an extended reversetranscription primer and locked nucleic acid modified PCR. Custom miRNAassays can be tested by running the assay on a dilution series ofchemically synthesized miRNA corresponding to the target sequence. Thispermits determination of the limit of detection and linear range ofquantitation of each assay. Furthermore, when used as a standard curve,these data permit an estimate of the absolute abundance of miRNAsmeasured in the samples. Amplification curves may optionally be checkedto verify that Ct values are assessed in the linear range of eachamplification plot. Typically, the linear range spans several orders ofmagnitude. For each candidate miRNA assayed, a chemically synthesizedversion of the miRNA can be obtained and analyzed in a dilution seriesto determine the limit of sensitivity of the assay, and the linear rangeof quantitation. Relative expression levels may be determined, forexample, according to the 2(−ΔΔ C(T)) Method, as described by Livak etah, Analysis of relative gene expression data using real-timequantitative PCR and the 2(−ΔΔ C(T)) Method. Methods (2001) December;25(4):402-8.

Rolling circle amplification is a DNA-polymerase driven reaction thatcan replicate circularized oligonucleotide probes with either linear orgeometric kinetics under isothermal conditions (see, for example,Lizardi et al., Nat. Gen. (1998) 19(3):225-232; Gusev et al, Am. J.Pathol. (2001) 159(1):63-69; Nallur et al, Nucleic Acids Res. (2001)29(23):E118). In the presence of two primers, one hybridizing to the (+)strand of DNA, and the other hybridizing to the (−) strand, a complexpattern of strand displacement results in the generation of over 10⁹copies of each DNA molecule in 90 minutes or less. Tandemly linkedcopies of a closed circle DNA molecule may be formed by using a singleprimer. The process can also be performed using a matrix-associated DNA.The template used for rolling circle amplification may be reversetranscribed. This method can be used as a highly sensitive indicator ofmiRNA sequence and expression level at very low miRNA concentrations(see, for example, Cheng et al., Angew Chem. Int. Ed. Engl. (2009)48(18):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91).

miRNAs quantification method may be performed by using stem-loop primersfor reverse transcription (RT) followed by a real-time TaqMan® probe.Typically, said method comprises a first step wherein the stem-loopprimers are annealed to miRNA targets and extended in the presence ofreverse transcriptase. Then miRNA-specific forward primer, TaqMan®probe, and reverse primer are used for PCR reactions. Quantitation ofmiRNAs is estimated based on measured CT values.

Many miRNA quantification assays are commercially available from Qiagen(S. A. Courtaboeuf, France), Exiqon (Vedbaek, Denmark) or AppliedBiosystems (Foster City, USA).

Expression level of miR-92a may be expressed as absolute expressionlevel or normalized expression level. Typically, expression levels arenormalized by correcting the absolute expression level of miR-92a bycomparing its expression to the expression of a mRNA that is not arelevant for determining patient having or at risk of having ordeveloping a rapidly progressive glomerulonephritis (RPGN), e.g., ahousekeeping mRNA that is constitutively expressed. Suitable mRNA fornormalization include housekeeping mRNAs such as the U6, U24, U48 andS18. This normalization allows the comparison of the expression level inone sample, e.g., a subject sample, to another sample, or betweensamples from different sources.

Nucleic acids exhibiting sequence complementarity or homology to themiRNA of interest herein find utility as hybridization probes oramplification primers. It is understood that such nucleic acids need notbe identical, but are typically at least about 80% identical to thehomologous region of comparable size, more preferably 85% identical andeven more preferably 90-95% identical. In certain embodiments, it willbe advantageous to use nucleic acids in combination with appropriatemeans, such as a detectable label, for detecting hybridization. A widevariety of appropriate indicators are known in the art including,fluorescent, radioactive, enzymatic or other ligands (e. g.avidin/biotin).

The probes and primers are “specific” to the miR-92a they hybridize to,i.e. they preferably hybridize under high stringency hybridizationconditions (corresponding to the highest melting temperature Tm, e.g.,50% formamide, 5× or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

miRNA may be detected using hybridization-based methods, including butnot limited to hybridization arrays (e.g., microarrays), NanoStringanalysis, Northern Blot analysis, branched DNA (bDNA) signalamplification, and in situ hybridization.

Microarrays can be used to measure the expression level of miR-92a.Microarrays can be fabricated using a variety of technologies, includingprinting with fine-pointed pins onto glass slides, photolithographyusing pre-made masks, photolithography using dynamic micromirrordevices, inkjet printing, or electrochemistry on microelectrode arrays.Also useful are microfluidic TaqMan Low-Density Arrays, which are basedon an array of microfluidic qRT-PCR reactions, as well as relatedmicrofluidic qRT-PCR based methods. In one example of microarraydetection, various oligonucleotides (e.g., 200+ 5′-amino-modified-C6oligos) corresponding to human sense miRNA sequences are spotted onthree-dimensional CodeLink slides (GE Health/Amersham Biosciences) at afinal concentration of about 20 μM and processed according tomanufacturer's recommendations. First strand cDNA synthesized from 20 μgTRIzol-purified total RNA is labeled with biotinylated ddUTP using theEnzo BioArray end labeling kit (Enzo Life Sciences Inc.). Hybridization,staining, and washing can be performed according to a modifiedAffymetrix Antisense genome array protocol. Axon B-4000 scanner andGene-Pix Pro 4.0 software or other suitable software can be used to scanimages. Non-positive spots after background subtraction, and outliersdetected by the ESD procedure, are removed. The resulting signalintensity values are normalized to per-chip median values and then usedto obtain geometric means and standard errors. miRNA signal can betransformed to log base 2, and a one-sample t test can be conducted.Independent hybridizations for each sample can be performed on chipswith miRNA spotted multiple times to increase the robustness of thedata.

Microarrays can be used for the expression profiling of miR-92a. Forexample, RNA can be extracted from the sample and, optionally, themiRNAs are size-selected from total RNA. Oligonucleotide linkers can beattached to the 5′ and 3′ ends of the miRNAs and the resulting ligationproducts are used as templates for an RT-PCR reaction. The sense strandPCR primer can have a fluorophore attached to its 5′ end, therebylabeling the sense strand of the PCR product. The PCR product isdenatured and then hybridized to the microarray. A PCR product, referredto as the target nucleic acid that is complementary to the correspondingmiRNA capture probe sequence on the array will hybridize, via basepairing, to the spot at which the, capture probes are affixed. The spotwill then fluoresce when excited using a microarray laser scanner. Thefluorescence intensity of each spot is then evaluated in terms of thenumber of copies of a particular miRNA, using a number of positive andnegative controls and array data normalization methods, which willresult in assessment of the level of expression of a particular miRNA.Total RNA containing the miRNA extracted from the sample can also beused directly without size-selection of the miRNAs. For example, the RNAcan be 3′ end labeled using T4 RNA ligase and a fluorophore-labeledshort RNA linker. Fluorophore-labeled miRNAs complementary to thecorresponding miRNA capture probe sequences on the array hybridize, viabase pairing, to the spot at which the capture probes are affixed. Thefluorescence intensity of each spot is then evaluated in terms of thenumber of copies of a particular miRNA, using a number of positive andnegative controls and array data normalization methods, which willresult in assessment of the level of expression of a particular miRNA.Several types of microarrays can be employed including, but not limitedto, spotted oligonucleotide microarrays, pre-fabricated oligonucleotidemicroarrays or spotted long oligonucleotide arrays.

Accordingly, the nucleic acid probes include one or more labels, forexample to permit detection of a target nucleic acid molecule using thedisclosed probes. In various applications, such as in situ hybridizationprocedures, a nucleic acid probe includes a label (e.g., a detectablelabel). A “detectable label” is a molecule or material that can be usedto produce a detectable signal that indicates the presence orconcentration of the probe (particularly the bound or hybridized probe)in a sample. Thus, a labeled nucleic acid molecule provides an indicatorof the presence or concentration of a target nucleic acid sequence(e.g., genomic target nucleic acid sequence) (to which the labeleduniquely specific nucleic acid molecule is bound or hybridized) in asample. A label associated with one or more nucleic acid molecules (suchas a probe generated by the disclosed methods) can be detected eitherdirectly or indirectly. A label can be detected by any known or yet tobe discovered mechanism including absorption, emission and/or scatteringof a photon (including radio frequency, microwave frequency, infraredfrequency, visible frequency and ultra-violet frequency photons).Detectable labels include colored, fluorescent, phosphorescent andluminescent molecules and materials, catalysts (such as enzymes) thatconvert one substance into another substance to provide a detectabledifference (such as by converting a colorless substance into a coloredsubstance or vice versa, or by producing a precipitate or increasingsample turbidity), haptens that can be detected by antibody bindinginteractions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules(or fluorochromes). Numerous fluorochromes are known to those of skillin the art, and can be selected, for example from Life Technologies(formerly Invitrogen), e.g., see, The Handbook-A Guide to FluorescentProbes and Labeling Technologies). Examples of particular fluorophoresthat can be attached (for example, chemically conjugated) to a nucleicacid molecule (such as a uniquely specific binding region) are providedin U.S. Pat. No. 5,866,366 to Nazarenko et al., such as4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS),N-(4-anilino-1-naphthyl)maleimide, antl1ranilamide, Brilliant Yellow,coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumarin 151);cyanosine; 4′,6-diarninidino-2-phenylindole (DAPI);5′,5″dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulforlic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6dicl1lorotriazin-2-yDarnino fluorescein (DTAF),2′7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC Q(RITC);2′,7′-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B,sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine101 (Texas Red); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA);tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);riboflavin; rosolic acid and terbium chelate derivatives. Other suitablefluorophores include thiol-reactive europium chelates which emit atapproximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27,1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, Lissamine™,diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein,4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No.5,800,996 to Lee et al.) and derivatives thereof. Other fluorophoresknown to those skilled in the art can also be used, for example thoseavailable from Life Technologies (Invitrogen; Molecular Probes (Eugene,Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, asdescribed in U.S. Pat. Nos. 5,696,157, 6,130,101 and 6,716,979), theBODIPY series of dyes (dipyrrometheneboron difluoride dyes, for exampleas described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782,5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an aminereactive derivative of the sulfonated pyrene described in U.S. Pat. No.5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent labelcan be a fluorescent nanoparticle, such as a semiconductor nanocrystal,e.g., a QUANTUM DOT™ (obtained, for example, from Life Technologies(QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.);see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649,138).Semiconductor nanocrystals are microscopic particles havingsize-dependent optical and/or electrical properties. When semiconductornanocrystals are illuminated with a primary energy source, a secondaryemission of energy occurs of a frequency that corresponds to the handgapof the semiconductor material used in the semiconductor nanocrystal.This emission can be detected as colored light of a specific wavelengthor fluorescence. Semiconductor nanocrystals with different spectralcharacteristics are described in e.g., U.S. Pat. No. 6,602,671.Semiconductor nanocrystals that can be coupled to a variety ofbiological molecules (including dNTPs and/or nucleic acids) orsubstrates by techniques described in, for example, Bruchez et al.,Science 281:20132016, 1998; Chan et al., Science 281:2016-2018, 1998;and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals ofvarious compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927,069;6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736;6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807;5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No.2003/0165951 as well as PCT Publication No. 99/26299 (published May 27,1999). Separate populations of semiconductor nanocrystals can beproduced that are identifiable based on their different spectralcharacteristics. For example, semiconductor nanocrystals can be producedthat emit light of different colors based on their composition, size orsize and composition. For example, quantum dots that emit light atdifferent wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mnemission wavelengths), which are suitable as fluorescent labels in theprobes disclosed herein are available from Life Technologies (Carlsbad,Calif.).

RT-PCR is typically carried out in a thermal cycler with the capacity toilluminate each sample with a beam of light of a specified wavelengthand detect the fluorescence emitted by the excited fluorophore. Thethermal cycler is also able to rapidly heat and chill samples, therebytaking advantage of the physicochemical properties of the nucleic acidsand thermal polymerase. The majority of the thermocyclers on the marketnow offer similar characteristics. Typically, thermocyclers involve aformat of glass capillaries, plastics tubes, 96-well plates or 384-wellsplates. The thermocylcer also involve a software analysis.

miR-92a can also be detected without amplification using the nCounterAnalysis System (NanoString Technologies, Seattle, Wash.). Thistechnology employs two nucleic acid-based probes that hybridize insolution (e.g., a reporter probe and a capture probe). Afterhybridization, excess probes are removed, and probe/target complexes areanalyzed in accordance with the manufacturer's protocol. nCounter miRNAassay kits are available from NanoString Technologies, which are capableof distinguishing between highly similar miRNAs with great specificity.The basis of the nCounter® Analysis system is the unique code assignedto each nucleic acid target to be assayed (International PatentApplication Publication No. WO 08/124847, U.S. Pat. No. 8,415,102 andGeiss et al. Nature Biotechnology. 2008. 26(3): 317-325; the contents ofwhich are each incorporated herein by reference in their entireties).The code is composed of an ordered series of colored fluorescent spotswhich create a unique barcode for each target to be assayed. A pair ofprobes is designed for each DNA or RNA target, a biotinylated captureprobe and a reporter probe carrying the fluorescent barcode. This systemis also referred to, herein, as the nanoreporter code system. Specificreporter and capture probes are synthesized for each target. Thereporter probe can comprise at a least a first label attachment regionto which are attached one or more label monomers that emit lightconstituting a first signal; at least a second label attachment region,which is non-over-lapping with the first label attachment region, towhich are attached one or more label monomers that emit lightconstituting a second signal; and a first target-specific sequence.Preferably, each sequence specific reporter probe comprises a targetspecific sequence capable of hybridizing to no more than one gene andoptionally comprises at least three, or at least four label attachmentregions, said attachment regions comprising one or more label monomersthat emit light, constituting at least a third signal, or at least afourth signal, respectively. The capture probe can comprise a secondtarget-specific sequence; and a first affinity tag. In some embodiments,the capture probe can also comprise one or more label attachmentregions. Preferably, the first target-specific sequence of the reporterprobe and the second target-specific sequence of the capture probehybridize to different regions of the same gene to be detected. Reporterand capture probes are all pooled into a single hybridization mixture,the “probe library”. The relative abundance of each target is measuredin a single multiplexed hybridization reaction. The method comprisescontacting the tumor sample with a probe library, such that the presenceof the target in the sample creates a probe pair-target complex. Thecomplex is then purified. More specifically, the sample is combined withthe probe library, and hybridization occurs in solution. Afterhybridization, the tripartite hybridized complexes (probe pairs andtarget) are purified in a two-step procedure using magnetic beads linkedto oligonucleotides complementary to universal sequences present on thecapture and reporter probes. This dual purification process allows thehybridization reaction to be driven to completion with a large excess oftarget-specific probes, as they are ultimately removed, and, thus, donot interfere with binding and imaging of the sample. All posthybridization steps are handled robotically on a custom liquid-handlingrobot (Prep Station, NanoString Technologies). Purified reactions aretypically deposited by the Prep Station into individual flow cells of asample cartridge, bound to a streptavidin-coated surface via the captureprobe, electrophoresed to elongate the reporter probes, and immobilized.After processing, the sample cartridge is transferred to a fullyautomated imaging and data collection device (Digital Analyzer,NanoString Technologies). The expression level of a target is measuredby imaging each sample and counting the number of times the code forthat target is detected. For each sample, typically 600 fields-of-view(FOV) are imaged (1376×1024 pixels) representing approximately 10 mm2 ofthe binding surface. Typical imaging density is 100-1200 countedreporters per field of view depending on the degree of multiplexing, theamount of sample input, and overall target abundance. Data is output insimple spreadsheet format listing the number of counts per target, persample. This system can be used along with nanoreporters. Additionaldisclosure regarding nanoreporters can be found in InternationalPublication No. WO 07/076129 and WO07/076132, and US Patent PublicationNo. 2010/0015607 and 2010/0261026, the contents of which areincorporated herein in their entireties. Further, the term nucleic acidprobes and nanoreporters can include the rationally designed (e.g.synthetic sequences) described in International Publication No. WO2010/019826 and US Patent Publication No. 2010/0047924, incorporatedherein by reference in its entirety.

Mass spectroscopy can be used to quantify miRNA using RNase mapping.Isolated RNAs can be enzymatically digested with RNA endonucleases(RNases) having high specificity (e.g., RNase T1, which cleaves at the3′-side of all unmodified guanosine residues) prior to their analysis byMS or tandem MS (MS/MS) approaches. The first approach developedutilized the on-line chromatographic separation of endonuclease digestsby reversed phase HPLC coupled directly to ESTMS. The presence ofposttranscriptional modifications can be revealed by mass shifts fromthose expected based upon the RNA sequence. Ions of anomalousmass/charge values can then be isolated for tandem MS sequencing tolocate the sequence placement of the posttranscriptionally modifiednucleoside. Matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) has also been used as an analytical approach forobtaining information about posttranscriptionally modified nucleosides.MALDI-based approaches can be differentiated from ESTbased approaches bythe separation step. In MALDI-MS, the mass spectrometer is used toseparate the miRNA. To analyze a limited quantity of intact miRNAs, asystem of capillary LC coupled with nanoESI-MS can be employed, by usinga linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL,Thermo Fisher Scientific) or a tandem-quadrupole time-of-flight massspectrometer (QSTAR® XL, Applied Biosystems) equipped with a custom-madenanospray ion source, a Nanovolume Valve (Valco Instruments), and asplitless nano HPLC system (DINa, KYA Technologies). Analyte/TEAA isloaded onto a nano-LC trap column, desalted, and then concentrated.Intact miRNAs are eluted from the trap column and directly injected intoa CI 8 capillary column, and chromatographed by RP-HPLC using a gradientof solvents of increasing polarity. The chromatographic eluent issprayed from a sprayer tip attached to the capillary column, using anionization voltage that allows ions to be scanned in the negativepolarity mode.

Additional methods for miRNA detection and measurement include, forexample, strand invasion assay (Third Wave Technologies, Inc.), surfaceplasmon resonance (SPR), cDNA, MTDNA (metallic DNA; AdvanceTechnologies, Saskatoon, SK), and single-molecule methods such as theone developed by US Genomics. miR-92a can be detected in a microarrayformat using a novel approach that combines a surface enzyme reactionwith nanoparticle-amplified SPR imaging (SPRI). The surface reaction ofpoly(A) polymerase creates poly(A) tails on miRNAs hybridized ontolocked nucleic acid (LNA) microarrays. DNA-modified nanoparticles arethen adsorbed onto the poly(A) tails and detected with SPRI. Thisultrasensitive nanoparticle-amplified SPRI methodology can be used formiRNA profiling at attamole levels. miRNA can also be detected usingbranched DNA (bDNA) signal amplification (see, for example, Urdea,Nature Biotechnology (1994), 12:926-928). miRNA assays based on bDNAsignal amplification are commercially available. One such assay is theQuantiGene® 2.0 miRNA Assay (Affymetrix, Santa Clara, Calif.). NorthernBlot and in situ hybridization may also be used to detect miRNAs.Suitable methods for performing Northern Blot and in situ hybridizationare known in the art. Advanced sequencing methods can likewise be usedas available. For example, miRNA can be detected using Illumina® NextGeneration Sequencing (e.g. Sequencing-By-Synthesis or TruSeq methods,using, for example, the HiSeq, HiScan, GenomeAnalyzer, or MiSeq systems(Illumina, Inc., San Diego, Calif.)). miRNA can also be detected usingIon Torrent Sequencing (Ion Torrent Systems, Inc., Gulliford, Conn.), orother suitable methods of semiconductor sequencing.

A further aspect of the invention relates to a method of preventing ortreating extracapillary glomerulonephritis, rapidly progressiveglomerulonephritis (RPGN) or collapsing glomerulopathy (CG) in a subjectin need thereof comprising the steps of:

i) providing a sample from a subject,

ii) measuring the expression level of miR-92a in the sample obtained atstep i),

iii) comparing said expression level measured in step ii) with acontrol, wherein high expression level of miR-92a is indicative ofsubject having or at risk of having or developing an extracapillaryglomerulonephritis, a rapidly progressive glomerulonephritis (RPGN) or acollapsing glomerulopathy (CG), and

iv) treating said subject having or at risk of having or developing anextracapillary glomerulonephritis, a rapidly progressiveglomerulonephritis (RPGN) or a collapsing glomerulopathy (CG) with acompound according to the invention and/or an extracapillaryglomerulonephritis treatment, a rapidly progressive glomerulonephritis(RPGN) treatment and/or a collapsing glomerulopathy (CG) treatment.

A further aspect of the invention relates to a method for monitoring theefficacy of a treatment for an extracapillary glomerulonephritis, arapidly progressive glomerulonephritis (RPGN) or a collapsingglomerulopathy (CG) in a subject in need thereof.

Methods of the invention can be applied for monitoring the treatment(e.g., drug compounds) of the subject. For example, the effectiveness ofan agent to affect the expression level of the miR-92a according to theinvention can be monitored during treatments of subjects receivingextracapillary glomerulonephritis treatments, rapidly progressiveglomerulonephritis (RPGN) treatments or collapsing glomerulopathy (CG)treatments.

The “rapidly progressive glomerulonephritis (RPGN) treatment” that isreferred to in the definition of step a) above relate to any type ofrapidly progressive glomerulonephritis (RPGN) therapy undergone by therapidly progressive glomerulonephritis (RPGN) subjects previously tocollecting the rapidly progressive glomerulonephritis (RPGN) tissuesamples, including cyclophosphamide, plasmapheresis, anti-CD20 antibody,mycophenolate mofetil and corticosteroids such as methylpredniso lone orprednisone.

The “collapsing glomerulopathy (CG) treatment” that is referred to inthe definition of step a) above relate to any type of collapsingglomerulopathy (CG) therapy undergone by the collapsing glomerulopathy(CG) subjects previously to collecting the CG tissue samples, includingsteroids or cyclosporine, angiotensin converting enzyme inhibitorsand/or angiotensin II receptor blockers, anti-HIV therapy, lipidlowering agents and mycophenolate mofetil.

Accordingly, the present invention relates to a method for monitoringthe treatment of subject affected with an extracapillaryglomerulonephritis, a rapidly progressive glomerulonephritis (RPGN) orcollapsing glomerulopathy (CG), said method comprising the stepsconsisting of:

i) diagnosis of extracapillary glomerulonephritis, rapidly progressiveglomerulonephritis (RPGN) or collapsing glomerulopathy (CG) before saidtreatment by performing the method of the invention

ii) diagnosis of extracapillary glomerulonephritis, rapidly progressiveglomerulonephritis (RPGN) or collapsing glomerulopathy (CG) after saidtreatment by performing the method of the invention

iii) and comparing the results determined a step i) with the resultsdetermined at step ii) wherein a difference between said results isindicative of the effectiveness of the treatment.

Oligonucleotide Sequences

>SEQ ID NO: 1 for hsa-mir-92a-1 MI0000093CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG >SEQ ID NO: 2 for hsa-miR-92a-1-5p MIMAT0004507AGGUUGGGAUCGGUUGCAAUGCU >SEQ ID NO: 3 for hsa-miR-92a-3p MIMAT0000092UAUUGCACUUGUCCCGGCCUGU >SEQ ID NO: 4 for hsa-mir-92a-2 MI0000094UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCCUGUGGAAGA >SEQ ID NO: 5 for hsa-miR-92a-2-5p MIMAT0004508GGGUGGGGAUUUGUUGCAUUAC >SEQ ID NO: 6 for hsa-miR-92a-3p MIMAT0000092UAUUGCACUUGUCCCGGCCUGU >SEQ ID NO: 7 for anti-miR-ctrlAAGGCAAGCUGACCCUGAAGUU >SEQ ID NO: 8 for anti-miR-92aCAGGCCGGGACAAGUGCAAUA

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: STAT3 activation in the glomerulus during RPGN in mice andhumans. Western blot analysis of phospho-STAT3 (Tyr705) and total STAT3expression in glomeruli extracts of control and mice challenged withanti-glomerular basement membrane nephrotoxic serum (NTS). Tubulin isused as loading control. Values are means sem from at least 6 mice. **P<0.01 versus control mice.

FIG. 2: STAT3 pharmacological blockade prevents renal destruction duringRPGN. (A) Western blot analysis of phospho-STAT3 (Tyr705) expression inrenal cortex extracts from NTS-injected mice treated with vehicle alone(NTS) and NTS-injected mice treated with Stattic, a STAT3 inhibitor,started in same time of NTS (NTS+Stattic). Tubulin is used as loadingcontrol. (B) Urinary albumin excretion rates and (C) blood urea nitrogenconcentration at day 10 after NTS injection in groups of mice as in A.(D) Proportion of crescentic glomeruli in groups of mice as in A. Valuesare means sem from at least 6 mice. * P<0.05 versus NTS-injected mice.

FIG. 3: Podocyte-specific deletion of Stat3 does not modeify glomerularmorphology and function. (A) Western blot analysis of total STAT3expression in primary podocyte culture from Pod-Stat3 WT and Pod-Stat3lox mice. Tubulin is used as loading control. (B) Albumin-to-creatinineratio and (C) blood urea nitrogen concentration in Pod-Stat3 WT andPod-Stat3 lox mice at baseline. Values are means sem (n=9 mice) **P<0.01 versus Pod-Stat3 WT mice.

FIG. 4: Stat3 inhibition reduces proliferative and migratory podocytephenotype in vitro. (A) Representative pictures and quantification ofpodocyte proliferation assay over 4 days from decapsulated glomeruli. (Band C) RT-PCR analysis of Ki67 (B) and PCNA (C) mRNA expression inprimary podocyte cultures with or without Stattic (2 μM) for 16 hours orfrom Pod-Stat3 lox mice. (D) Representative images of wound assayshowing migration within 12 hours of podocytes incubated with or without2 μM Stattic or from Pod-Stat3 lox mice. Scale bars 100 μm. Values aremeans sem (n=6 mice). * P<0.05, *** P<0.001 versus untreated podocytes.

FIG. 5: Deletion of Stat3 in podocyte protects against renal dysfunctionduring experimental RPGN. (A) Proportion of crescentic glomeruli inPod-Stat3 WT and Pod-Stat3 lox mice at day 10 after NTS injection. (Band C) Albuminuria (B) and blood urea nitrogen (C) concentrations inNTS-challenged Pod-Stat3 WT and Pod-Stat3 lox mice. (D) Number ofpodocyte foot processes per 10 μm glomerular basement membrane (GBM)length. Values are means sem from at least 5 mice. ** P<0.01; ***P<0.001 versus NTS-challenged Pod-Stat3 WT mice.

FIG. 6: Podocyte-specific deletion of Stat3 decreases glomerularepithelial cells proliferation in RPGN. (A and B) RT-PCR analysis ofKi67 (A) and PCNA (B) mRNA expression in isolated glomeruli fromunchallenged or NTS-injected Pod-Stat3 WT and Pod-Stat3 lox mice. Valuesare means sem from at least 6 mice. ** P<0.01; *** P<0.001 versus nonchallenged mice and #P<0.001 versus NTS-challenged Pod-Stat3 WT mice.

FIG. 7: Upstream pathways of Stat3 activation in podocytes. (A) Westernblot analysis and quantification of phospho-EGFR (Tyr1068) andphospho-Stat3 (Tyr705) expression in kidney cortex from control orNTS-challenged mice with or without erlotinib (10 mg/kg/day), a specificinhibitor of EGFR. Tubulin is used as loading control. Values are meanssem from at least 5 mice per condition. * P<0.05 versus unchallengedmice (control). (B) Western blot analysis and quantification ofphospho-EGFR (Tyr1068) and phospho-STAT3 (Tyr705) on primary culture ofpodocytes after addition of AG1478 (EGFR kinase inhibitor), anti-mIL-6monoclonal antibody or HB-EGF for 16 hours. Values are means sem from atleast 5 western blot. * P<0.05 versus untreated podocytes (control).

FIG. 8: Upregulation of miR-92a expression with RPGN in mouse and humankidneys. (A) RT-qPCR for miR-92a normalized to U6 and relative tocontrol in the renal cortex from normal mouse kidneys or NTS-challengedmice or NTS-challenged mice with Stattic or NTS-challenged s mice.Values are means sem (n=4 per group). * P<0.05 vs. control, † P<0.05 vs.NTS alone. (B) Relative expression of hsa-mir-92a in kidney biopsiesfrom individuals diagnosed with ANCA-associated vasculitis and RPGN andnormal kidney samples. Values are means sem (n=4 per group). * P<0.05vs. normal.

FIG. 9: Inhibition of miR-92a in cultured podocytes decreaseproliferation with p57 upregulation. (A) RT-qPCR for miR-92a normalizedto U6 and relative to control in podocytes transfected with ananti-miR-control (anti-miR-ctrl) or an anti-miR-92a. (B) Representativepictures and quantification of podocyte proliferation assay over 4 daysfrom decapsulated mouse glomeruli. (C) RT-PCR analysis of Ki67 mRNAexpression in primary podocyte cultures with or without inhibition ofmiR-92a. (D) Western blot analysis and quantification of p57 proteinexpression in primary cultured podocytes with or without inhibition ofmiR-92a. Tubulin is used as loading control. Values are means sem (n=4per group). * P<0.05, ** P<0.01 and *** P<0.001 versus control podocytes(control).

FIG. 10: Only the miR-92a member of the miR-17-92 microRNA cluster isupregulated in injured glomeruli. RT-qPCR expression analysis formiR-92a, miR-17, miR-18a, miR-19a and miR-20a normalized to U6 andrelative to control in freshly isolated glomeruli from normal mice(control), NTS-challenged mice (NTS), NTS-challenged mice treated withanti-miR-control (anti-miR-ctrl) and NTS-challenged mice treated withanti-miR-92a (anti-miR-92a) after 10 days.

FIG. 11: Silencing miR-92a decreases kidney injury in mouse model ofRPGN.

(A) and blood urea nitrogen concentrations (B) in normal mice (control),NTS-challenged mice (NTS), NTS-challenged mice treated withanti-miR-control (anti-miR-ctrl) and NTS-challenged mice treated withanti-miR-92a (anti-miR-92a) 10 days after NTS injection. Values aremeans sem (n=4 per group). * P<0.05 versus control, # P<0.05 vs. NTSalone.

FIG. 12: miR-92a in vivo silencing prevents and abolishes RPGNdevelopment.

(A) Study design of the in vivo antagomir experiment. (B) RelativemiR-92a expression in dynebeads isolated glomeruli from normal mice(control), NTS-challenged mice (NTS), NTS-challenged mice treated withanti-miR-control (anti-miR-ctrl) and NTS-challenged mice treated withanti-miR-92a (anti-miR-92a) after 10 days. All values are normalized toU6 and are relative to control. Values are means±sem (n=5 per group). *p<0.05 vs. control, # p<0.05 vs. NTS alone. (C) Urinary albuminexcretion rates at day 4 (before antagomir injection) and at day 10after NTS injection. Scale bars 10 μm. (D) Blood urea nitrogenconcentration at day 10 after NTS injection in control or NTS-challengedmice. Values are means±sem (n=10 mice per group). (E) Quantification ofp57-positive cells per glomerular section in mice described in (a).Values are means±sem (n=10 per group). * p<0.05, # p<0.05 vs. NTS alone(NTS).

EXAMPLES Example 1

Material & Methods

Animals

Podocyte-specific disruption of Stat3 mice were generated by crossingpodocin-Cre positive mice (63) with Stat3 floxed mice (64) on C57BL6/Jbackground. Their age-matched littermates with no deletion of Stat3 inany cells are considered as controls. Pharmacological inhibition ofStat3 was achieved with Stattic, a non-peptidic small molecule thatselectively inhibits activation, dimerization and nuclear translocationof STAT3 (65). Stattic (25 mg/kg) was administered in i.p. way everyother day for 10 days. 10 weeks old 129S2/SvPasCr male mice wererandomly treated with Stattic and were compared to vehicle-treated(DMSO) littermates.

Experiments were conducted according to the French veterinary guidelinesand those formulated by the European Community for experimental animaluse (L358-86/609EEC), and were approved by the Institut National de laSanté et de la Recherche Médicale and local University Research EthicsCommittee (file 12-62, Comité d'Ethique en matière d′ExpérimentationAnimale, Paris Descartes).

Human Tissues

Formalin-fixed, paraffin-embedded renal tissue specimens obtained at theHôpital Européen Georges Pompidou, Assistance Publique-Hopitaux deParis, Paris, France, were included in this study. Human tissue was usedafter approval by, and following the guidelines of, the local EthicsCommittee. Renal biopsy cases with sufficient tissue forimmunohistochemical evaluation after completion of diagnostic workupwere included. Normal adult human kidney tissue was obtained fromkidneys surgically excised because of the presence of a localizedneoplasm.

Induction of Crescentic Glomerulonephritis

The glomerulonephritis was induced on male mice (10-12 weeks of age) byintravenous injection of 15 μL of sheep anti-glomerular basementmembrane (GBM) nephrotoxic serum (NTS), which was diluted with 850 μL ofsterile phosphate buffer solution. Serum injections were repeated twice(on days 2 and 3) at 6 μL/g of body weight and 7 μL/g respectively.

Biochemical Measurements in Blood and Urine

Urinary creatinine and blood urea nitrogen (BUN) concentrations wereanalyzed by a standard colorimetric method (Olympus AU400) at theBiochemistry Laboratory of Institut Claude Bernard (IFR2, Faculté deMédecine Paris Diderot). Urinary albumin excretion was measured using aspecific ELISA assay for quantitative determination of albumin in mouseurine (CellTrend GmbH).

Glomeruli Isolation and In Vitro Assays in Cultured Podocytes

Mouse kidneys were extracted, minced, and digested in 2 mg/mLcollagenase I solution (Gibco) in RPMI 1640 (Invitrogen) at 37° C. for 3minutes, then filtered through a 70-μm cell strainer and one morethrough a 40-μm cell strainer. The homogenate was centrifuged at 720 gfor 6 minutes and cells plated. Isolated glomeruli were then collectedin Phosphosafe extraction buffer (Novagen) for protein extraction or inRLT extraction buffer (Qiagen) for total RNA extraction. For podocyteprimary culture, freshly isolated glomeruli were plated in 6-platedishes in RPMI 1640 (Invitrogen) supplemented by 10% Fetal Calf Serum(Biowest) and 1% penicillin-streptomycin (Invitrogen). The outgrowth ofpodocytes started between days 2 and 3. Podocyte outgrowth area wasquantified at day 4 using ImageJ software.

After 4 days of primary culture, podocytes were trypsinized then platedinto μ-Dish 35 mm high with Culture-Insert (Ibidi). Ibidi's woundinginserts were used for cell migration studies. The coverage of the 500-μmgap was assessed after 12 hours of culture and podocyte migration areawas quantified using ImageJ software. The effects of HB-EGF (10 ng/mL,Preprotech) or AG1478 (1 μM, Calbiochem) or anti-mIL-6 monoclonalantibody MP5-20F3 (10 μg/mL, eBiosciences) or Stattic (2 μM, Calbiochem)on differentiated podocytes was applied during 16 hours. Afterstimulation, podocytes were scrapped in Phosphosafe buffer for proteinextraction or in RLT buffer for total RNA extraction.

Histology

Kidneys were harvested and fixed in 4% formal. Paraffin-embeddedsections (5 μm thick) were stained by Masson's trichrome to evaluatekidney morphology and determine proportion of crescentic glomeruli by ablinded examination on at least 50 glomeruli per section.

Immunohistochemistry and Immunofluorescence

Deparaffinized kidney sections were incubated for 30 minutes at 95° C.in the target retrieval solution (S1699, Dako), then in peroxidaseblocking reagent (S2001, Dako), blocked in PBS containing 5% BSA andimmunostained against phospho-STAT3 (Tyr705) (Cell Signaling Technology)and phospho-STAT3 (Tyr705) (Millipore), STAT3 (Cell SignalingTechnology), podocalyxin (R&D systems), Ki-67 (Abcam) and p57 (SantaCruz Technology). For phospho-STAT3, Ki-67 and p57, specific stainingwas revealed using Histofine reagents (Nichirei Biosciences), whichcontained anti-rabbit (414341F) or anti-goat (414161F) immune-peroxidasepolymer for mouse tissue sections. For STAT3 and podocalyxinimmunofluorescence, primary antibody incubation was followed by asecondary rabbit anti-goat IgG AF488-conjugated antibody (Invitrogen,1:400) and a secondary rabbit anti-goat IgG AF594-conjugated antibody(Invitrogen, 1:400) respectively. Podocyte culture cells wereimmunostained against podocin (ab50339 Abcam), nephrin (ab58968 Abcam),WT1 (ab15249 Abcam) or p57 (Santa Cruz Biotechnology). The nuclei werestained using DAPI. Images were obtained with an Axioimager Z1 apotome(Zeiss).

Transmission Electron Microscopy Procedure

Small pieces of renal cortex were fixed in 4% glutaraldehyde, postfixedin 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sectionswere counterstained with uranyl acetate and examined in a JEOL 1011transmission electron microscope with Digital Micrograph software foracquisition. Measurements of podocyte foot process, per 10 μm ofglomerular basement membrane (GBM), were made on the resultingphotographs by counting manually. Results for 100 μm of GBM wereaveraged.

Western Blot Analysis

After extraction from glomeruli or podocytes with lysis buffer, proteinswere quantified by BCA protein assay kit (iNtRON Biotechnology). Sampleswere resolved on 4-12% Bis-Tris Criterion XT gels (Bio-Rad) thentransferred to a polyvinylidene difloride membrane. Membranes wereincubated with the appropriate primary antibodies: rabbitanti-phospho-EGFR (Tyr1068) (Cell Signaling Technology), rabbitanti-phospho-STAT3 (Tyr705) (Cell Signaling Technology), goat anti-p57(Santa Cruz Biotechnology). Protein loading was monitored by using therat anti-tubulin antibody (Abcam). Secondary antibodies were donkey-antirabbit HRP and donkey-anti rabbit HRP (GE Healthcare Life Sciences).Antigens were revealed by enhanced chemiluminescence (Supersignal WestPico, Pierce) and detected on a LAS-4000 imaging system (Fuji).Densitometric analysis with ImageJ software was used for quantification.

Real-Time PCR

Total RNA extraction of mice glomeruli was performed using an RneasyMinikit (Qiagen) and reverse transcribed into cDNA using the QuantitectReverse Transcription kit (Qiagen) according to the manufacturer'sprotocol. cDNA and standard were amplified in Maxima SYBR Green/Rox qPCRmix (Fermentas) on an ABI PRISM thermo cycler. The comparative method ofrelative quantification (2-ΔΔCT) was used to calculate the expressionlevel of each target gene, normalized to GAPDH. The oligonucleotidesequences are available upon request. The data are presented as the foldchange in gene expression.

For miRNA expression analysis, RNA from kidney cortex were prepared withTrizol (Life Technologies) according to manufacturer's instructions.miRNA expression was determined using Taqman miRNA assay (Lifetechnologies) according to manufacturer's protocols. U6 snRNA was usedas endogenous control.

miR-92a In Situ Hybridization

In situ hybridization was performed as previously described by Bonaueret al (66). Briefly, 5 μm-thick kidney paraffin embedded sections werecut and fixed in PFA 4% for 10 min. Then sections were washed with 1×PBSand then acetylated for 10 min. After washes, sections were incubatedwith protein kinase K (Sigma-Aldrich) for 10 min at 37° C. Afterwashings, sections were incubated with hybridization buffer for 5 h atroom temperature. miRNA probes (miR-92a probe double-DIG labeled LNAprobes, Exiqon, final concentration 20 nM) was mixed with denaturationbuffer and added to the sections followed by incubation over night at56° C. U6snRNA probe (3′-DIG labeled LAN, probe, Exiqon) was used at 10nM final concentration and as a positive control. The day after,sections were washed in successive decreasing SSC buffers for 5 min at56° C. (5×1 time, 1×2 times, 0.2×3 times) and then washed. Afterincubation for 1 hour in blocking solution (B1 solution+3% fetal calfserum+0.1% Tween-20), sections were incubated with anti-DIG AP antibody(Roche; 1:2000) overnight at 4° C. After washings, sections wereincubated with NBT/BCIP (Promega) in NTMT+levamisole (0.2 mM/L) for 48hours in the dark at RT. NBT/BCIP was changed every 12 hrs. Afterwards,slides were fixed in PFA 4% for 30 min and mounted with Fluoprepmounting medium (Biomerieux).

In Vivo miR-92a Inhibition in Wild-Type Mice

For preventive strategy, antagomiR treatment (12 mg/kg) started 3 daysbefore NTS injection. AntagomiRs (VBC biotech, Vienna) were delivered byretro-orbital i.v. injections under brief anesthesia. Second and thirdinjections were performed on days 1 and 3 with NTS injection. Forcurative strategy, antagomir were injected on days 4, 5 and 8 after NTS.A scramble antagomiR (Antagomir-Control) was used as control. Thesequences were obtained from previously published manuscript (66). Inthe miR-92 antagomir and control antagomir, the 2′O RNA base aremethylated followed by first two bases and last 3 bases arephosphorothiated to increase the stability of antagomir fromdegradation. In addition, a cholesterol-TEG was added at the 3′ for easyentry of antagomir into the cells. AntagomiR-Control (SEQ ID NO: 7,anti-miR-ctrl): 5′-AAGGCAAGCUGACCCUGAAGUU-3′ and antagomiR-92a (SEQ IDNO: 8, anti-miR-92a): 5′-CAGGCCGGGACAAGUGCAAUA-3′ as shown to beeffective after in vivo administration in kidneys (34, 67).Saline-treated mice were used as control of the scramble antagomiR.

miR-92a In Vitro Modulation

MicroRNA-92a inhibition was achieved in vitro by transfecting primaryculture podocytes with anti-miR-92a inhibitor using Hiperfecttransfection reagent (Qiagen). Anti-miR-Control#1 was used as control(All from Ambion, 50 nM).

Statistical Analyses

All values are expressed as means+SEM. Statistical analyses werecalculated using GraphPad Prism software (La Jolla, Calif.). Comparisonbetween two groups was performed by using Mann-Whitney t test.Comparison between multiple groups was performed by using one-way ANOVAfollowed by Tukey post test. Values of P<0.05 were consideredsignificant.

Results

Activation of STAT3 in Glomeruli During Crescentic RPGN in Mice andHumans

Recently, the inventors found that EGFR activation in podocytes isinvolved in glomerular injury and renal failure during RPGN (8). Todecipher the signaling pathway implicated in RPGN, the inventorsinvestigated downstream signals in the EGFR pathway. STAT3 activationwas studied in kidney harvested 10 days after injection of anti-GBMnephrotoxic serum (NTS) into mice. First, phosphorylation of STAT3 ontyrosine 705 appeared in freshly isolated glomeruli from NTS-treatedthan control animals (FIG. 1). To determine which cell type evinced anactivation of STAT3, the inventors performed immunohistochemistry onkidney biopsies from NTS-challenged and control mice. In non-treatedmice, STAT3 is exclusively phosphorylated in tubular cells. A stainingfor phospho-STAT3 (Tyr705) appeared in glomerular cells as podocytes ofNTS-treated mice. To evaluate the relevance of this finding to humandisease, the inventors performed immunohistochemical staining of kidneysections from 6 patients diagnosed with RPGN complicatingAnti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis. All sectionsof kidney biopsies showed a staining for phospho-STAT3 in podocytes andin crescents.

STAT3 Pharmacological Blockade Prevents Renal Destruction During RPGN

To determine whether blockade of STAT3 activation could represent apossible therapeutic complement for treatment of RPGN as found with EFGRkinase inhibitors (8), the inventors injected concomitantly NTS andStattic, a STAT3 inhibitor (8) to WT mice with the 129S2 geneticbackground that is markedly prone to crescent formation and glomerulardamage upon NTS administration. Stattic administration significantlyreduced STAT3 (Tyr705) phosphorylation in glomeruli after 10 days ofsevere experimental RPGN (FIG. 2A). STAT3 inhibition induced a trend todecreased albuminuria (FIG. 2B), significantly alleviated the rise inblood urea nitrogen (BUN) concentrations by 50% (FIG. 2C) and reducedthe proportion of crescentic glomeruli (FIG. 2D). Overall,administration of systemic STAT3 inhibitor had marked effects on renaldamage, inflammation and renal failure in this severe experimental modelof RPGN.

Deletion of Stat3 Prevents Switch in Podocyte Phenotype

To abolish STAT3 expression specifically in podocyte, the inventors bredmice in order to obtain mice with Stat3 floxed and podocyte-specific Creexpression (Pod). To verify STAT3 deficiency in podocytes, the inventorsperformed immunofluorescence detection of total STAT3 protein andpodocalyxin, as cell surface marker of podocyte, in kidney sections ofPod-Stat3 lox and Pod-Stat3 WT mice. Double staining of sectionsrevealed marked constitutive expression of STAT3 in podocytes ofglomeruli from Pod-Stat3 WT mice. The inventors show that STAT3 stainingis nearly absent in podocytes from Pod-Stat3 lox mice. Deletion of STAT3was also confirmed in primary culture of podocyte from Pod-Stat3 lox andPod-Stat3 WT mice. Western blot analysis showed a significant decreasein STAT3 expression in cultured primary podocytes (change, −88%) (FIG.3A). It should be noticed that after isolation from kidney, less than 5%of cells were other glomerular cells than podocytes (WT1/podocinnegative cells).

Pod-Stat3 lox mice showed no abnormalities in glomerular morphology,urinary albumin excretion and renal function estimated by BUN (FIGS. 3Band C). Thus, Stat3 alleles deletion in podocytes did not disturb renalphenotype at baseline. the inventors next isolated glomeruli fromPod-Stat3 lox and Pod-Stat3 WT mice to compare podocyte proliferationand migration, hallmarks of podocyte dedifferentiation and crescentformation. Thus, as an in vitro assay for podocyte crescent formation,the inventors measured outgrowth of podocytes from isolated decapsulatedmouse glomeruli. When cultured for 7 days, 95% of cells still exhibitpodocin, nephrin and WT1 expression. Podocyte outgrowth area wassignificantly reduced around glomeruli isolated from Pod-Stat3 lox miceor in Pod-Stat3 WT podocytes treated with STAT3 inhibitor (FIG. 4A).Likewise, genetic or pharmacological alteration of STAT3 markedlyblunted Ki67 and PCNA expression in primary podocytes (FIGS. 4B and C).By contrast, STAT3 inhibition or podocyte-specific deletion of Stat3displayed little or no effect on the migratory phenotype (FIG. 4D).

Podocyte-Specific Deletion of Stat3 Protects Against Renal DestructionDuring RPGN

To study in vivo the relative contribution of STAT3 in podocytes in thedevelopment of inflammatory glomerular injury, the inventors challengedPod-Stat3 lox mice with NTS. As expected, Pod-Stat3 WT mice exhibitedcrescent formation, and renal dysfunction. However, Pod-Stat3 lox micedisplayed a marked 60% decrease in crescent formation evaluated inMasson's trichrome stained sections (FIG. 5A). Furthermore, thishistological observation was associated with preserved renal function inPod-Stat3 lox animals only, as shown by reduced urinary albumin tocreatinine ratio (FIG. 5B) and normal BUN (FIG. 5C) concentrations.Podocyte-specific deletion of Stat3 also attenuated ultrastructuralalterations of podocytes and full loss of interdigitating foot processpattern. The counted foot process per 10 μm of GBM is 5-fold higher inPod-Stat3 lox mice, suggesting a conservation of podocyte ultrastructureand differentiated cytoskeletal architecture (FIG. 5D). To decipher therole of STAT3 on podocyte phenotype in vivo, the inventors performedreal-time PCR for Ki67 and PCNA mRNA expression in freshly isolatedglomeruli from unchallenged and NTS-challenged Pod-Stat3 lox andPod-Stat3 WT mice. Administration of NTS upregulated PCNA (change,+130%) (FIG. 6A) and Ki67 (change, +128%) (FIG. 6B) mRNA levels infreshly isolated glomeruli from Pod-Stat3 WT mice. NTS-induced Ki67glomerular expression was found in podocytes and parietal epithelialcells (PECs) only. This effect of NTS on ki67 and PCNA mRNA levels wasalmost completely abolished by Stat3 deletion in podocyte (FIGS. 6A, andB). Taken together these findings suggest that Pod-Stat3 lox mice wereprotected from NTS-induced crescent formation and loss of renalfunction.

Upstream Inducers of STAT3 Activation in Podocytes.

Known stimulators of STAT3 signaling are ligand-mediated activation ofgp130 receptor and epidermal growth factor (EGF) receptor (18-20) byIL-6 (21-23), IL-10 (24-26) and EGFR ligands such as HB-EGF (27),respectively. The inventors first focused on the HB-EGF/EGFR pathwaybecause activation of this pathway in podocytes was found to aggravateRPGN (8). Assessment of p-EGFR and p-STAT3 expression in freshlyisolated glomeruli from NTS-challenged and normal mice with or with noEGFR antagonist provided evidence for EGFR-dependent modulation of STAT3activation in vivo (FIG. 7A).

To examine whether IL-6 and HB-EGF could activate STAT3 pathway directlyin podocytes, the inventors blocked IL-6 and EGFR in cultured primarypodocytes and then performed western blotting to determine STAT3phosphorylation (Tyr705), which is a marker of activation of STAT3signaling. Cultured podocytes display features of dedifferentiation withacquired proliferative capacity and constitutive EGFR and STAT3activation. The inventors found that both specific kinase inhibitor ofEGFR, AG1478 and anti-mIL-6 monoclonal antibody blunted STAT3phosphorylation (FIG. 7B). Furthermore, these data indicate that IL-6receptor (IL-6R) and EGFR pathway are tonically activated by autocrinesynthesis of ligands by activated podocytes. Taken together, these datasuggest that both EGFR and IL-6R may stimulate STAT3 pathway inactivated podocytes in vitro and in vivo during crescentic RPGN.

STAT3 Activity Upregulates miR-92a in Podocytes During RPGN.

Because of the potent action of STAT3 on podocyte phenotype, theinventors suspected that STAT3 activation may target a large set ofgenes through modulation of microRNAs. In particular, STAT3 has beenshown to activate several microRNAs in various diseases (15, 28).Because of the presence of highly conserved STAT3-binding site in thepromoter region of the miR-17/92 gene, the inventors have studied theexpression of miR-17/92a in experimental crescentic GN. The inventorsfound a widespread upregulation of miR-92a throughout kidney fromNTS-challenged nephritic Pod-Stat3 WT, particularly in glomerular cells.MicroRNA-92a expression was not increased in Stattic-treated Pod-Stat3WT animals nor in Pod-Stat3 lox mice. The upregulation shown by miR-92ain situ hybridization on kidney sections was confirmed by RT-qPCR (FIG.8A). Consistent with findings on mouse model, miR-92a was significantlyupregulated in kidney from 4 patients diagnosed with crescenticglomerulonephritis caused by ANCA-associated vasculitides compared tocontrols subjects (FIG. 8B).

miR-92a Inhibition In Vitro Decreases Podocyte Proliferation

To decipher the role of miR-92a on podocyte function, the inventorsinhibited miR-92a expression in primary culture of podocytes (change,−71%) (FIG. 9A). Anti-miR-92a did not affect STAT3 protein level,suggesting that STAT3 is an upstream regulator of miR-92a (data notshown). Delivery of anti-miR-92a to podocytes caused a decrease inoutgrowth (FIG. 9B) and in Ki67 mRNA level (FIG. 9C), compared tocontrol cells or transfected with an anti-miR control. Therefore,miR-92a seems to be involved in the regulation of podocyteproliferation. Thus, the inventors next searched for potential targetsof miR-92a in target prediction algorithms as miRanda, miRWalk andTargetScan. The inventors focused on genes that have been previouslyrelated to podocyte proliferation. Among potential candidates, theinventors found a member of the Cip/Kip family, thep57/Kip2/Cyclin-dependent kinase inhibitor 1C, a tight-binding inhibitorof several G1 cyclin/Cdk complexes and a negative regulator of cellproliferation (29, 30). Overexpressing p57 leads to G1 phase cell cyclearrest and different studies have shown that p57 is constitutivelyexpressed in mature podocytes (31, 32) as the inventors also found, anddecrease in p57 protein expression during glomerular disease isassociated with an increase in podocyte proliferation (33). Theinventors studied the p57 protein expression by western blot andimmunofluorescence in anti-miR-92a transfected podocytes (FIG. 9D). Inthis condition, our results show an increase in p57 expression,correlating with a decrease in podocyte proliferation.

Inhibition of miR-92a in Mice Ameliorates Glomerular Injury.

Chemically engineered oligonucleotides, termed ‘antagomirs’, areefficient and specific silencers of endogenous miRNAs in mice. It waspreviously demonstrated that tiny, miRNA-inhibiting antagomirs(anti-miR) are taken up into the kidney cortex after intravenousinjection into mice (34).

The inventors then tested the feasibility of this anti-miR-92a strategyto prevent the development of RPGN. The inventors induced RPGN in miceand injected anti-miR-92a. Anti-miR-92a injections allowed for specificinhibition of miR-92a expression in isolated glomeruli during RPGN (FIG.10) without modify levels of others miR of 17-92 cluster. Compared witheither control anti-miR (NTS+anti-miR-ctrl) or vehicle only (NTS),anti-miR92a administration led to less glomerular injury, shown by lessglomerular crescents formation (−44%, P<0.05 vs NTS+anti-miR-ctrl) and astrong decrease in urinary albumin excretion (FIG. 11A) and kidneydysfunction (FIG. 11B).

To determine whether miR-92a inhibition could have an effect on p57expression in vivo, the inventors performed immunostaining for p57 onkidney sections of mice injected with anti-miR-92a. Induction ofglomerulonephritis induced a decrease in p57 expression in glomerulithat was rescued by anti-miR-92a administration (FIG. 11C).

The inventors validated these results in curative strategy by antagomirinjection on days 4, 5 and 8 after NTS (FIG. 12 A-E).

Discussion

RPGN with extracapillary proliferation of epithelial glomerular cells isa major clinical problem because it does not fully respond toimmunosuppressive therapy and leads to chronic renal failure. As RPGNinvolves primarily the podocytes reaction to immune injury, deeperinsights into the stabilization mechanisms of podocytes are important,and the question of a potential role of regulatory miRNAs arises.However, the role of specific miRNAs, to the best of our knowledge, hasnot been addressed. Comparing signaling cascade profiling in primarypodocytes, in a mouse model of RPGN and random human kidney samplesdiagnosed with crescentic GN, the inventors identified pathwayspromoting miR-92a upregulation, in particular EGFR-mediated STAT3activation in podocytes. The studies presented here systematicallydemonstrate the involvement of miR-92a in the deleterious response toimmune injury that leads to glomerular destruction and functionaldemise. Our studies further identified a key miR-92a target asp57/Kip2/Cyclin-dependent kinase inhibitor 1C that is involved in cellcycle regulation and control of quiescent state of podocytes.

The miR-92a is part of the miR-17-92 cluster. The miR-17-92 cluster is apolycistrionic miRNA that encodes 6 miRNAs (35). Mice deficient formiR-17-92 die rapidly after birth with described cardiac and lungabnormalities. Thus, this study suggested a physiological importancerole of this cluster in development. Interestingly, miR-17-92 wasdescribed as an oncogenic miRNA cluster, first as involved in B-celllymphoma. However, other studies have reported contrasting roles forthese miRNAs, both as a cluster and as individually miRNAs. Among themembers in the cluster, miR-92a is the least characterized subunit. Therole of specific miRNAs and in particular miR-92a, to the best of ourknowledge, has not been addressed in crescentic RPGN.

Here, for the first time the inventors provided data about miR-92aupregulation in this severe kidney disease. Given that miRNAs areconserved, with regard to both evolution and function, our observationof a common crescentic miRNA expression pattern in murine RPGN and humancrescentic lesions is likely to have significant pathogenic, diagnostic,and/or therapeutic implications in human RPGNs.

A novel finding of our study is that we identify STAT3 as a keyregulator in podocytes that activates miR-92a in glomeruli duringcrescentic RPGN. Phosphorylated forms of STAT3 (p-STAT3) are found atlow level in normal glomeruli (36). Expression of the JAK/Stat3 pathwayin non podocyte cells has been involved in several models of glomerulardiseases. P-STAT3 involvement in inflammatory response of culturesmesangial cells in vitro has been demonstrated (37, 38) and followed bystudies of experimental mesangial diseases. STAT3 activation was againfound in mesangial cells and influenced the progression of diabeticnephropathy (39, 40) and of Thy1 glomerulonephritis (41) Inhibition ofJAK2 signaling ameliorated the course of adriamycin nephropathy in mouse(42). However, the dramatic changes in podocyte phenotype occurring increscentic RPGN are absent in these models, and no conclusion regardinga potential role of STAT3 in RPGN could be drawn from these studies.Interestingly, evidence that STAT3 activation modulates podocytephenotype has been recently brought in the specific setting ofHIV-associated nephropathy (HIVAN). Although the mechanisms of RPGNdiffer from those of HIVAN in many points, these two diseases share somesimilarities in podocyte changes. Indeed, podocytes loose their specificmarkers and proliferate in both RPGN and HIVAN (4, 5, 43, 44). Specificmechanisms are at play promoting HIVAN, with evidence that the HIV-1protein Nef activates the STAT3 and MAPK1,2 pathways, fostering podocyteproliferation and dedifferentiation (45). In HIV-1 transgenic mice(Tg26), renal injury was alleviated by suppression of STAT3 limited topodocytes (46). Very recently, these authors published similar findingsthan ours, using a mouse model of podocyte-specific deletion of Stat3 inan accelerated model of NTS-induced RPGN (47). Altogether, thesefindings indicate that STAT3 orchestration of podocyte phenotype may bea general paradigm for proliferative extracapillary diseases and ourstudy provides additional insights and relevance to human disease withproof of principle for potential therapy since systemic STAT3 blockadewith Stattic mimicked the protective action of podocyte targeted Stat3gene deletion and prevented renal failure. Observation that STAT3blockade by Stattic inhibited proliferation and migration of culturedprimary podocytes indicate that STAT3 stimulates alteration of podocytephenotype such as seen in RPGN. The potent anti-proliferative actionprovided by STAT3 deficiency was further demonstrated in vivo sinceStat3 specific deletion in podocytes resulted in reduced expression ofthe proliferation markers PCNA and Ki67 in renal cortex and in podocytesand consistently prevented crescent formation during RPGN.

Another salient result in our study was the observation that STAT3activation was induced not only in mouse but also in human RPGNassociated with ANCA-associated vasculitis. The inventors foundexpression of phosphorylated STAT3 in crescentic glomeruli of patientswith RPGN whereas no or faint staining for phosphorylated STAT3 wasdetected in normal glomeruli. Although phosphorylated STAT3 was mainlydetected in podocytes and glomerular crescents, positive immunostainingwas also observed in some parietal epithelial cells. Therefore, STAT3may be also involved in the proliferation of parietal epithelial cells,which is known to contribute to crescent formation (3).

The inventors did not investigate whether STAT3 activation was involvedin the loss of podocyte differentiation markers like podocin, nephrinand synaptopodin that typically occurs along with de novo expression ofproliferation markers in RPGN (5, 6). In experimental HIVAN, Tg26 micewith reduced STAT3 activity showed preservation of synaptopodin,podocin, and WT-1 expression. Podocyte-specific deletion of STAT3resulted in similar prevention of podocyte dedifferentiation in Tg26mice (46). A limitation of our study may be that the inventors did notexamine the effect of STAT3 activation on vascular endothelial growthfactor (VEGF) expression in our model. STAT3 was shown to mediateHIV-induced VEGF expression in podocytes, which may represent a criticalstep in the development of HIVAN (48, 49). However, a role for increasedVEGF expression in glomerular injury seems less likely in RPGN than inHIVAN. Indeed, podocyte-specific overexpression of VEGF in mouse led tocollapsing glomerulopathy, recapitulating the phenotype of HIVAN, butnot RPGN (50). In addition, blockade of VEGF did not improve but ratherworsened the course of RPGN in rat (51). At last, our transcriptomeanalysis of isolated glomeruli show diminished VEGFA and receptors mRNAexpression in nephritic mice when compared to unchallenged mice (datanot shown). Thus, altogether, these data suggest that STAT3 activationis part of distinct signaling networks in podocytes in RPGN and inHIVAN.

The inventors further found pathways upstream of STAT3 activation inpodocytes. The inventors recently demonstrated in a mouse model ofanti-glomerular basement membrane (anti-GBM) glomerulonephritis that thelack of EGFR ligand HB-EGF or pharmacological blockade of EGFR orgenetic deletion of Egfr alleles in podocytes prevented crescentformation and markedly alleviated the course of RPGN (8). The evidencedemonstrating functional or direct association between EGFR and STAT3 isbased primarily on work done in cell lines expressing high levels ofEGFR, such as A431, and head and neck squamous cell carcinoma (HNSCC)cells (11, 19, 27, 52). Given that EGFR-mediated intracellular eventscan be transduced through activation of STAT3 (10, 11, 19, 20, 53), theinventors investigated whether inhibition of EGFR could influence STAT3activation in RPGN. Selective EGFR kinase inhibitor erlotinibefficiently blocked EGFR phosphorylation in the kidney cortex and alsoblunted STAT3 phosphorylation in podocytes. In the case of RPGN, EGFRautocrine/paracrine activation is promoted by depression of the Hbegfgene in podocytes and parietal epithelial cells (8, 54). Interestingly,autocrine activation of STAT3 by HB-EGF has been reported in breastcancer lines (27). However, despite apparent complete inhibition ofEGFR, the phosphorylated form of STAT3 remained low but detectable,indicating that STAT3 activation was not entirely mediated through EGFRsignaling. In line with these results in vivo, the inventors observed inprimary cultures of podocytes that STAT3 phosphorylation was enhanced byHB-EGF, and was decreased by the EGFR kinase inhibitor AG1478.Furthermore, neutralization of IL-6 also blunted STAT3 phosphorylation.This suggests that cultured primary podocyte acquiring an ‘activated’phenotype display autocrine activation of both EGFR and IL-6R pathwaysleading to downstream STAT3 activation with proliferation.

Therefore, it appears that EGFR and IL-6 signaling are significantmechanisms driving STAT3 activation in RPGN and in cultured podocyteswith upregulation of miR-92a expression. In line with this finding, acase of anti-neutrophil cytoplasmic antibody (ANCA)—associatedcrescentic RPGN has been successfully treated using anti-IL-6 receptorantibody (55).

The inventors also identified a relevant miR-92a target for podocyteproliferation. In contrast to immature podocytes, which proliferateduring glomerular development in utero, differentiated podocytes have aterminally differentiated quiescent phenotype (56). The maintenance of adifferentiated phenotype is required for podocytes to perform theirspecialized functions (56). Using three independent target predictionalgorithms, the inventors identified p57 as a miR-92a target. The CDKinhibitor p57 regulates cell proliferation and differentiation (57). P57is typically expressed in differentiated and postmitotic nonrenal cells,and Shankland et al. and others have shown that there is de novoexpression of p57 in podocytes during glomerulogenesis that coincideswith p27 expression and with podocyte acquisition of a terminallydifferentiated phenotype (33, 58). Loss of p57 expression in podocyteswas early recognized a feature of proliferative glomerular diseases (5,59-61) although the mechanism remained elusive. The inventors cannotexclude that other miR-92a targets such as PTEN (62) or Reck (16) couldbe involved in podocyte dedifferentiation process. Nevertheless, theinventors confirmed that a decrease or absence of p57 immunostaining wasassociated with damage and proliferative podocyte phenotype inexperimental RPGN. Importantly, a decrease in the level of p57 and acorresponding de novo expression of Ki67 coincided with podocyteproliferation in vitro and in vivo as observed with administration ofantagomir that specifically silence miR-92a and effectively reversed thedeleterious effects of miR-92a in kidney injuries.

Our combined results obtained from in vitro experiments, mouse modelsand human tissues indicate that increased expression of miR-92a caninitiate a cascade of podocyte-destabilizing molecular events startingwith the downregulation of p57 and proliferation. Moreover, specificblockade of miR-92a in vivo by an antagomir markedly reducedproteinuria, crescent formation and renal failure. Although thistreatment showed a preventive effect in our mouse model, it remains tobe seen whether this holds true for human RPGN as well.

In summary, the inventors provide evidence for a new pathogenicmechanism for RPGN that is driven by STAT3-mediated upregulation ofmiR-92a and decrease in p57, unlocking podocyte ability to proliferatewith ensuing proteinuria, destruction of the glomerular filtrationbarrier and declining renal function.

Example 2 High Expression of miR-92a in Human Kidneys with RPGN

To evaluate the clinical relevance of the results obtained in the mousemodel, the inventors analyzed miR-92a expression by in situhybridization and RT-PCR in paraffin-embedded tissue of renal biopsiesfrom patients with RPGN and control patients with non proliferativeglomerulopathies (Table 1). MiR-92a labeling was weak and restricted tothe endothelium in control human kidneys (non-crescentic glomerulardisease). However, miR-92a was significantly upregulated in kidneybiopsies from patients with RPGN, regardless of its etiology. In situhybridization revealed the expression of miR-92a in glomerular cells ofpatients with RPGN, particularly in podocytes and crescents and to alesser extent in parietal epithelial cells. RT-qPCR analysis revealedthat the expression of miR-92a was three to five fold higher in samplesfrom patients with RPGN of various etiologies including stage III and IVlupus nephritis (Lup), microscopic polyangiitis (MPA) and granulomatosiswith polyangiitis (GPA) than those from patients diagnosed withnoncrescentic glomerulopathies, other chronic proteinuric glomerulardiseases including minimal change disease (MCD) and membranousnephropathy (MM). The inventors observed a similar pattern of miR-92aexpression in all kidney samples from patients with RPGN regardless ofimmunological etiology. These results show that miR-92a is highlyabundant, sometimes in a sustained fashion, in conditions associatedwith glomerular epithelial cell proliferation and crescent formation.

TABLE 1 Patients' clinical details Gender Age Diagnosis RelapseTreatment F 29 Minimal Change Disease Relapse Corticosteroids F 30Minimal Change Disease Relapse No treatment M 66 Minimal Change DiseaseFirst episode No treatment F 31 Minimal Change Disease First episode Notreatment F 30 Membranous Nephropathy First episode No treatment M 46Membranous Nephropathy First episode No treatment M 50 MembranousNephropathy First episode No treatment M 62 Micro-polyangiitis Firstepisode No treatment MPO-ANCA-positive M 78 Micro-polyangiitis Firstepisode Corticosteroids PR3-ANCA-positive M 52 Micro-polyangiitis Firstepisode No treatment MPO-ANCA-positive F 46 Micro-polyangiitis Firstepisode No treatment MPO-ANCA-positive M 47 Micro-polyangiitis Firstepisode No treatment MPO-ANCA-positive F 58 Micro-polyangiitis Firstepisode No treatment MPO-ANCA-positive M 44 Micro-polyangiitis Firstepisode No treatment ANCA-positive M 68 Granulomatosis with Firstepisode Corticosteroids polyangiitis M 71 Granulomatosis with Firstepisode No treatment polyangiitis M 55 Granulomatosis with RelapseCorticosteroids polyangiitis F 35 Lupus nephritis (class IV) RelapseCorticosteroids F 27 Lupus nephritis (class IV) Relapse CorticosteroidsM 17 Lupus nephritis (class IV) First episode No treatment F 41 Lupusnephritis (class III) Relapse Corticosteroids F 25 Lupus nephritis(class III) First episode No treatment

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. A method of preventing or treatingextracapillary glomerulonephritis in a subject in need thereof,comprising the step of administering to said subject a miR-92a inhibitorcompound, wherein said miR-92a inhibitor compound is a nucleic acid thathybridizes with miR-92a.
 2. The method according to claim 1 wherein saidextracapillary glomerulonephritis is rapidly progressiveglomerulonephritis.
 3. The method according to claim 1 wherein saidextracapillary glomerulonephritis is collapsing glomerulopathy.
 4. Themethod according to claim 1 wherein said miR-92a inhibitor compound isselected from the group consisting of double-stranded RNA, antagomirs,antisense nucleic acids and enzymatic RNA molecules.
 5. The method ofclaim 1 wherein the miR-92a inhibitor compound is encoded by a vectorthat is administered to the subject.
 6. A method for preventing ortreating extracapillary glomerulonephritis in a subject, comprising thestep of administering to said subject a vector; and a miR-92a inhibitorcompound associated with said vector, wherein said miR-92a inhibitorcompound is a nucleic acid that hybridizes with miR-92a.